Hepatitis c virus codon optimized non-structural ns3/4a fusion gene

ABSTRACT

Aspects of the present invention relate to the discovery of a novel hepatitis C virus (HCV) isolate. Embodiments include HCV peptides, nucleic acids encoding said HCV peptides, antibodies directed to said peptides, compositions containing said nucleic acids and peptides, as well as methods of making and using the aforementioned compositions including, but not limited to, diagnostics and medicaments for the treatment and prevention of HCV infection.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of and claims the benefit of priorityto U.S. patent application Ser. No. 12/649,216, filed Dec. 29, 2009,which is a continuation of and claims the benefit of priority to U.S.patent application Ser. No. 12/001,735, filed Dec. 11, 2007, which is acontinuation of and claims the benefit of priority to U.S. patentapplication Ser. No. 11/043,808, filed Jan. 25, 2005, which is acontinuation of and claims the benefit of priority to U.S. patentapplication Ser. No. 10/307,047, filed Nov. 26, 2002, which iscontinuation-in-part of U.S. patent application Ser. No. 09/930,591,filed Aug. 15, 2001, and is a continuation-in-part of U.S. patentapplication Ser. No. 09/929,955, filed Aug. 15, 2001 and said U.S.patent application Ser. No. 09/930,591, filed Aug. 15, 2001, claims thebenefit of priority to U.S. Provisional Patent Application No.60/225,767, filed Aug. 17, 2000 and claims the benefit of priority toU.S. Provisional Patent Application No. 60/229,175, filed Aug. 29, 2000and said U.S. patent application Ser. No. 09/929,955 claims the benefitof priority to U.S. Provisional Patent Application No. 60/225,767, filedAug. 17, 2000 and claims the benefit of priority to U.S. ProvisionalPatent Application No. 60/229,175, filed Aug. 29, 2000. Accordingly, thepresent application claims priority to all of the aforementionedapplications and provisional applications and the disclosure of eachapplication. The contents of each application and provisionalapplication are hereby expressly incorporated by reference in theirentirety.

Reference to Sequence Listing

The present application is being filed along with a Sequence Listing inelectronic format. The Sequence Listing is provided as a file entitledTRIPEP.28CPC1C4.TXT, created Aug. 27, 2010, which is 85 KB in size. Theinformation in the electronic format of the Sequence Listing isincorporated herein by reference in its entirety.

FIELD OF THE INVENTION

Aspects of the present invention relate to the discovery of a novelhepatitis C virus (HCV) isolate. Embodiments include HCV peptides,nucleic acids encoding said HCV peptides, antibodies directed to saidpeptides, compositions containing said nucleic acids and peptides, aswell as methods of making and using the aforementioned compositionsincluding, but not limited to, diagnostics and medicaments for thetreatment and prevention of HCV infection.

BACKGROUND OF THE INVENTION

Viruses are intracellular parasites that require the biochemicalmachinery of a host cell for replication and propagation. All virusparticles contain some genetic information that encodes viral structuralproteins and enzymes. The genetic material may be DNA or RNA, in double-or single stranded form. (Virology, Fields ed., third edition,Lippencott-Raven publishers, pp 72-83 (1996)). The viral nucleic acid issurrounded by a coat of proteins called the capsid. (Id.) In someviruses the capsid is surrounded by an additional layer comprised of alipid membrane, referred to as the envelope. (Id. at 83-95).

The typical viral life cycle begins with infection of a host cellthrough attachment of the virus particle to a cell surface receptor andinternalization of the viral capsid. (Id. at 103). Accordingly, a virus'host range is limited to cells that express an appropriate cell surfacereceptor. Once internalized, the virus particle is disassembled and itsnucleic acid is transcribed, translated or replicated. (Id.) At thispoint, the virus may undergo lytic replication, where new virusparticles are formed and released from the infected cell. (Id. at105-11). The Influenza virus is a typical example of a virus thatundergoes lytic replication immediately upon infection of a host cell.(Id. at 1369-85).

Alternatively, a virus may enter a latent phase, referred to aslysogeny, where the genome is replicated but few if any viral proteinsare actually expressed and viral particles are not formed. (Id. at219-29). Herpesviruses such as the Epstein-Barr Virus are typicalexamples of viruses that establish latent infection in the host cells.(Id. at 229-34). Eventually, in order for the virus to spread, it mustexit lysogeny and enter the lytic phase. The viral particles that arereleased during the lytic phase infect other cells of the sameindividual or can be transmitted to another individual where a newinfection is established.

Since the viral life cycle comprises both an intracellular andextracellular phase, both the humoral and cell-mediated immune defensesystems are important for combating viral infections. (Id. at 467-73).Antibodies directed against viral proteins may block the virusparticle's interaction with its cellular receptor or otherwise interferewith the internalization or release processes. (Id. at 471). An antibodycapable of interfering with the viral life cycle is referred to as aneutralizing antibody.

During intracellular replication, viral proteins, which are foreign tothe host cell, are produced and some of these proteins are digested bycellular proteases after coupling to a Major Histocompatibility Complex(MEW) molecule presented on the surface of the infected cell. (Id. at350-58). Thus, the infected cell is recognized by T-lymphocytes,macrophages or NK-cells and killed before the virus replicates andspreads to adjacent cells. (Id. at 468-70). In addition, the presence ofviral nucleic acids, most notably as double-stranded RNA, triggers theinfected cell to shut down its translation machinery and to produceantiviral signaling molecules known as interferons. (Id. at 376-79).

Viruses have evolved various means of evading the immune defense systemof the host, however. By establishing latency (i.e., lysogeny), forexample, the virus does not enter the lytic phase and avoids the humoralimmune defense system. (Id. at 224). During the latent phase, few viralproteins are produced and infected cells have only a minimal ability topresent evidence to surrounding lymphocytes and macrophages of theirinfected state. (Id. at 225-26). Additionally, some viral proteins, mostnotably those produced during latency, evolve polypeptide sequences thatcannot be efficiently presented to the cell mediated immune defensesystem. (Levitskaya et al., Nature 375:685-88 (1995)). Finally, someviruses may actively interfere with the immune response of the infectedhost, for instance by preventing surface expression of MEW molecules(Fruh et al., J. Mol. Med. 75:18-27 (1997)), or by disrupting interferonsignaling (Fortunato et al., Trends Microbiol. 8:111-19 (2000)).

Particularly evasive are the hepatitis viruses, which are not classifiedas a family but are grouped based on their ability to infect cells ofthe liver. Hepatitis C Virus (HCV) belongs to the Flaviviridae family ofsingle-stranded RNA viruses. (Virology, supra, pp 945-51). The HCVgenome is approximately 9.6 kb in length, and encodes at least tenpolypeptides. (Kato, Microb. Comp. Genomics, 5:129-151 (2000)). Thegenomic RNA is translated into one single polyprotein that issubsequently cleaved by viral and cellular proteases to yield thefunctional polypeptides. (Id.) The polyprotein is cleaved to threestructural proteins (core protein, E1 and E2), to p7 of unknownfunction, and to six non-structural (NS) proteins (NS2, NS3, NS4A/B,NS5A/B). (Id.) NS3 encodes a serine protease that is responsible forsome of the proteolytic events required for virus maturation (Kwong etal., Antiviral Res., 41:67-84 (1999)) and NS4A acts as a co-factor forthe NS3 protease. (Id.) NS3 further displays NTPase activity, andpossesses RNA helicase activity in vitro. (Kwong et al., Curr. Top.Microbiol. Immunol., 242:171-96 (2000)).

HCV infection typically progresses from an acute to a chronic phase.(Virology, supra, pp 1041-47). Acute infection is characterized by highviral replication and high viral load in liver tissue and peripheralblood. (Id. at 1041-42.) The acute infection is cleared by the patient'simmune defense system in roughly 15% of the infected individuals; in theother 85% the virus establishes a chronic, persistent infection.(Lawrence, Adv. Intern. Med., 45:65-105 (2000)). During the chronicphase replication takes place in the liver, and some virus can bedetected in peripheral blood. (Virology, supra, pp 1042).

Essential to the establishment of a persistent infection is theevolution of strategies for evading the host's immune defense system.HCV, as a single stranded RNA virus, displays a high mutation rate inthe replication and transcription of its genome. (Id. at 1046). Thus, ithas been noted that the antibodies produced during the lytic phaseseldom neutralize virus strains produced during chronic infection. (Id.)Although it appears HCV is not interfering with antigen processing andpresentation on MHC-I molecules, the viral NS5A protein may be involvedin repression of interferon signaling through inhibition of the PKRprotein kinase. (Tan et al., Virology, 284:1-12 (2001)).

The infected host mounts both a humoral and a cellular immune responseagainst the HCV virus but in most cases the response fails to preventestablishment of the chronic disease. Following the acute phase, theinfected patient produces antiviral antibodies including neutralizingantibodies to the envelope proteins E1 and E2. (Id. at 1045). Thisantibody response is sustained during chronic infection. (Id.) Inchronically infected patients, the liver is also infiltrated by bothCD8+ and CD4+ lymphocytes. (Id. at 1044-45). Additionally, infectedpatients produce interferons as an early response to the viralinfection. (Id. at 1045). It is likely that the vigor of the initialimmune response against the infection determines whether the virus willbe cleared or whether the infection will progress to a chronic phase.(Pape et al., J. Viral. Hepat., 6 Supp. 1:36-40 (1999)). Despite theefforts of others, the need for efficient immunogens and medicaments forthe prevention and treatment of HCV infection is manifest.

SUMMARY OF THE INVENTION

A new HCV isolate was discovered. A novel NS3/4A fragment of the HCVgenome was cloned and sequenced from a patient infected with HCV (SEQ.ID. NO.: 1). This sequence was found to be only 93% homologous to themost closely related HCV sequence. Embodiments comprise, consist, orconsist essentially of this peptide (SEQ. ID. NO.: 2) or fragmentsthereof containing any number of consecutive amino acids between atleast 3-50 amino acids of SEQ. ID. NO.: 2 (e.g., 3, 4, 6, 8, 10, 12, 15,20, 25, 30, 35, 40, 45, or 50 consecutive amino acids), nucleic acidsencoding these molecules, vectors having said nucleic acids, and cellshaving said vectors, nucleic acids, or peptides. The NS3/4A nucleicacid, fragments thereof and corresponding peptides were found to beimmunogenic. Accordingly, preferred embodiments include vaccinecompositions and immunogen preparations comprising, consisting of, orconsisting essentially of the HCV peptide of SEQ. ID. NO.: 2 orfragments thereof (e.g., SEQ. ID. NOs.: 14 and 15) containing any numberof consecutive amino acids between at least 3-50 amino acids of SEQ. ID.NO.: 2 (e.g., 3, 4, 6, 8, 10, 12, 15, 20, 25, 30, 35, 40, 45, or 50consecutive amino acids) or a nucleic acid encoding said peptide orfragments.

Mutants of the NS3/4A peptide were also created and were found to beimmunogenic. Some mutants are truncated versions of the NS3/4A peptide(e.g., SEQ. ID. NOs.: 12 and 13) and others lack a proteolytic cleavagesite (e.g., SEQ. ID. NOs.: 3-11). These peptides (e.g., SEQ. ID. NOs.:3-13) and fragments thereof containing any number of consecutive aminoacids between at least 3-50 amino acids (e.g., 3, 4, 6, 8, 10, 12, 15,20, 25, 30, 35, 40, 45, or 50 consecutive amino acids) of any one ofSEQ. ID. NOs.: 3-13 (e.g., SEQ. ID. NOs.: 15-26), nucleic acids encodingthese molecules, vectors having said nucleic acids, and cells havingsaid vectors, nucleic acids, or peptides are embodiments of theinvention. A particularly preferred embodiment is a vaccine compositionor immunogen preparation comprising, consisting of, or consistingessentially of at least one HCV peptide of SEQ. ID. NOs.: 3-11 or afragment thereof containing any number of consecutive amino acidsbetween at least 3-50 amino acids (e.g., 3, 4, 6, 8, 10, 12, 15, 20, 25,30, 35, 40, 45, or 50 consecutive amino acids) of any one of SEQ. ID.NOs.: 3-11 (e.g., SEQ. ID. NOs.: 16-26) or a nucleic acid encoding saidpeptides or fragments.

Additional embodiments include a NS3/4a encoding nucleic acid orcorresponding peptide, which comprise a sequence that was optimized forcodons most frequently used in humans. The nucleic acid sequence of thecodon-optimized NS3/4A nucleic acid sequence is provided in SEQ. ID.NO.: 35, whereas the peptide encoded by said nucleic acid sequence isprovided in SEQ. ID. NO.: 36. This nucleic acid and corresponding NS3/4Apeptide do not correspond to any known HCV sequence or genome. Thecodon-optimized NS3/4A encoding nucleic acid was found to be only 79%homologous, within the region of nucleotide positions 3417-5475, toHCV-1 and contained a total of 433 different nucleotides. The NS3/4Apeptide encoded by the codon-optimized nucleic acid sequence was only98% homologous to HCV-1 and contained a total of 15 different aminoacids. The codon optimized nucleic acid was found to generate a higherexpression level of NS3 and was found to be more immunogenic, withrespect to both humoral and cellular responses, as compared to thenative NS3/4A gene.

Accordingly, aspects of the present invention include compositions thatcomprise, consist, or consist essentially of the nucleic acid sequenceprovided by the sequence of SEQ. ID. NO.: 35 and/or the peptide sequenceprovided by the sequence of SEQ. ID. NO.: 36. Preferred embodiments, forexample, include compositions that comprise, consist or consistessentially of any number of consecutive nucleotides between at least12-2112 nucleotides of SEQ. ID. NO.: 35 or a complement thereof (e.g.,12-15, 15-20, 20-30, 30-50, 50-100, 100-200, 200-500, 500-1000,1000-1500, 1500-2079, or 1500-2112 consecutive nucleotides). Preferredembodiments also include compositions that comprise, consist or consistessentially of any number of consecutive nucleotides between at least12-2112 nucleotides of SEQ. ID. NO.: 35 or a complement thereof (e.g.,at least 3, 4, 6, 8, 10, 12, 15, 20, 30, 40, 50, 60, 70, 80, 90, or 100consecutive amino acids of SEQ. ID. NO.: 35). Additional embodimentsinclude nucleic acids that comprise, consist, or consist essentially ofa sequence that encodes SEQ. ID. NO.: 36 or a fragment thereof, that is,any number of consecutive amino acids between at least 3-50 amino acidsof SEQ. ID. NO.: 36 (e.g., 3, 4, 6, 8, 10, 12, 15, 20, 25, 30, 35, 40,45, or 50 consecutive amino acids). Still more embodiments includepeptides that comprises, consist, or consist essentially of the sequenceof SEQ. ID. NO.: 36 or a fragment thereof, that is, any number ofconsecutive amino acids between at least 3-50 amino acids of SEQ. ID.NO.: 36 (e.g., 3, 4, 6, 8, 10, 12, 15, 20, 25, 30, 35, 40, 45, or 50consecutive amino acids).

Methods of making and using the compositions described herein are alsoprovided. In addition to methods of making the embodied nucleic acidsand peptides, other embodiments include methods of making immunogensand/or vaccine compositions that can be used to treat or prevent HCVinfection. Some methods are practiced, for example, by mixing anadjuvant with a peptide or nucleic acid antigen (e.g., an HCV peptide orHCV nucleic acid), as described above, so as to formulate a singlecomposition (e.g., a vaccine composition). Preferred methods involve themixing of ribavirin with an HCV gene or antigen disclosed herein.

Preferred methods of using the compositions described herein involveproviding an animal in need of an immune response to HCV with asufficient amount of one or more of the nucleic acid or peptideembodiments described herein. By one approach, for example, an animal inneed of an immune response to HCV (e.g., an animal at risk or alreadyinfected with HCV, such as a human) is identified and said animal isprovided an amount of NS3/4A (SEQ. ID. NO.: 2 or SEQ. ID. NO.: 36), amutant NS3/4A (SEQ. ID. NOs.: 3-13), a fragment thereof (e.g., SEQ. ID.NOs.: 14-26) or a nucleic acid encoding said molecules that is effectiveto enhance or facilitate an immune response to the hepatitis viralantigen. Additional methods are practiced by identifying an animal inneed of a potent immune response to HCV and providing said animal acomposition comprising a peptide comprising an antigen or epitopepresent on SEQ. ID. NOs.: 2-27 or SEQ. ID. NO.: 36 or a nucleic acidencoding said peptides. Particularly preferred methods involve theidentification of an animal in need of an immune response to HCV andproviding said animal a composition comprising an amount of HCV antigen(e.g., NS3/4A (SEQ. ID. NO.: 2 or SEQ. ID. NO.: 36)), mutant NS3/4A(SEQ. ID. NOs.: 3-13), a fragment thereof containing any number ofconsecutive amino acids between at least 3-50 amino acids (e.g., 3, 4,6, 8, 10, 12, 15, 20, 25, 30, 35, 40, 45, or 50 consecutive amino acids)of SEQ. ID. NO.: 2 or SEQ. ID. NO.: 36 (e.g., SEQ. ID. NOs.: 14-26) or anucleic acid encoding one or more of these molecules that is sufficientto enhance or facilitate an immune response to said antigen. In someembodiments, the composition described above also contains an amount ofribavirin that provides an adjuvant effect.

In still more embodiments, for example, a gene gun is used to administeran HCV nucleic acid described herein (e.g., SEQ. ID. NO.: 35 or fragmentthereof, as described above) to a mammalian subject in need of an immuneresponse to HCV. In some embodiments, an amount of ribavirin is mixedwith the DNA immunogen prior to delivery with the gene gun. In otherembodiments, the DNA immunogen is provided by gene gun shortly before orafter administration of ribavirin at or near the same site of DNAinoculation.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graph showing the antibody titer in H-2^(d) mice against NS3as a function of time after the first intra muscular immunization.Diamonds denote antibody titer in mice immunized with NS3/4A-pVAX andsquares denote antibody titer in mice immunized with NS3-pVAX.

FIG. 2 shows the in vivo protection conferred by one gene gunimmunization of NS3/4A-pVAX1 (4 μg) or MSLF1-pVAX1 (4 μg). Mice wereimmunized with the respective plasmid and 14 days later the mice werechallenged with an NS3/4A expressing SP2/0 cell line (approximately 10⁶cells/mouse). Tumor size was then measured through the skin dailyfollowing day 6 post-challenge and the data plotted.

FIG. 3 shows the in vivo protection conferred by two gene gunimmunizations of NS3/4A-pVAX1 (4 μg) or MSLF1-pVAX1 (4 μg). Mice wereimmunized with the respective plasmid at weeks zero and week four and,14 days after the last immunization, the mice were challenged with anNS3/4A expressing SP2/0 cell line (approximately 10⁶ cells/mouse). Tumorsize was then measured through the skin daily following day 6post-challenge and the data plotted.

FIG. 4 shows the in vivo protection conferred by three gene gunimmunizations of NS3/4A-pVAX1 (4 μg) or MSLF1-pVAX1 (4 μg). Mice wereimmunized with the respective plasmid at weeks zero, week four, and weekeight and, 14 days after the last immunization, the mice were challengedwith an NS3/4A expressing SP2/0 cell line (approximately 10⁶cells/mouse). Tumor size was then measured through the skin dailyfollowing day 6 post-challenge and the data plotted.

FIG. 5A is a graph showing the percentage of specific CTL-mediated lysisof SP2/0 target cells as a function of the effector to target ratio.Phosphate Buffered Saline (PBS) was used as a control immunogen.

FIG. 5B is a graph showing the percentage specific CTL-mediated lysis ofSP2/0 target cells as a function of the effector to target ratio.Plasmid NS3/4A-pVAX was used as the immunogen.

FIG. 6A is a graph showing the response of naive splenic T cells thatwere stimulated with peptide coated RMA-S cells. The naive splenic Tcells were obtained from C57/BL6 mice.

FIG. 6B is a graph showing the response of splenic T cells that wererestimulated with peptide coated RMA-S cells. The splenic T cells wereobtained from C57/BL6 mice that were provided a single 4 μg dose ofMSLF1-pVAX1.

FIG. 6C is a graph showing the response of splenic T cells that wererestimulated with peptide coated RMA-S cells. The splenic T cells wereobtained from C57/BL6 mice that were provided a single 4 μg dose ofNS3/4A-pVAX1.

FIG. 6D is a graph showing the response of naive splenic T cells thatwere stimulated with peptide coated RMA-S cells. The naive splenic Tcells were obtained from C57/BL6 mice.

FIG. 6E is a graph showing the response of splenic T cells that wererestimulated with peptide coated RMA-S cells. The splenic T cells wereobtained from C57/BL6 mice that were provided two 4 μg doses ofMSLF1-pVAX1.

FIG. 6F is a graph showing the response of splenic T cells that wererestimulated with peptide coated RMA-S cells. The splenic T cells wereobtained from C57/BL6 mice that were provided two 4 μg doses ofNS3/4A-pVAX1.

FIG. 6G is a graph showing the response of naive splenic T cells thatwere stimulated with NS3/4A expressing EL-4 cells. The naive splenic Tcells were obtained from C57/BL6 mice.

FIG. 6H is a graph showing the response of splenic T cells that wererestimulated with NS3/4A expressing EL-4 cells. The splenic T cells wereobtained from C57/BL6 mice that were provided a single 4 μg dose ofMSLF1-pVAX1.

FIG. 6I is a graph showing the response of splenic T cells that wererestimulated with NS3/4A expressing EL-4 cells. The splenic T cells wereobtained from C57/BL6 mice that were provided a single 4 μg dose ofNS3/4A-pVAX1.

FIG. 6J is a graph showing the response of naive splenic T cells thatwere stimulated with NS3/4A expressing EL-4 cells. The naive splenic Tcells were obtained from C57/BL6 mice.

FIG. 6K is a graph showing the response of splenic T cells that wererestimulated with NS3/4A expressing EL-4 cells. The splenic T cells wereobtained from C57/BL6 mice that were provided two 4 μg doses ofMSLF1-pVAX1.

FIG. 6L is a graph showing the response of splenic T cells that wererestimulated with NS3/4A expressing EL-4 cells. The splenic T cells wereobtained from C57/BL6 mice that were provided two 4 μg doses ofNS3/4A-pVAX1.

FIG. 7 is a graph showing the humoral response to 10 and 100 μgrecombinant Hepatitis C virus (HCV) non structural 3 protein (NS3), asdetermined by mean end point titres, when a single dose of 1 mg ofribavirin was co-administered.

FIG. 8 is a graph showing the humoral response to 20 μg recombinantHepatitis C virus (HCV) non structural 3 protein (NS3), as determined bymean end point titres, when a single dose of 0.1, 1.0, or 10 mg ofribavirin was co-administered.

FIG. 9 is a graph showing the effects of a single dose of 1 mg ribavirinon NS3-specific lymph node proliferative responses, as determined by invitro recall responses.

DETAILED DESCRIPTION OF THE INVENTION

A novel nucleic acid and protein corresponding to the NS3/4A domain ofHCV was cloned from a patient infected with HCV (SEQ. ID. NO.: 1). AGenebank search revealed that the cloned sequence had the greatesthomology to HCV sequences but was only 93% homologous to the closest HCVrelative (accession no AJ 278830). This novel peptide (SEQ. ID. NO.: 2)and fragments thereof (e.g., SEQ. ID. NOs.: 14 and 15) that are anynumber of consecutive amino acids between at least 3-50 (e.g., 3, 4, 6,8, 10, 12, 15, 20, 25, 30, 35, 40, 45, or 50 amino acids in length),nucleic acids encoding these molecules, vectors having said nucleicacids, and cells having said vectors, nucleic acids, or peptides areembodiments of the invention. It was also discovered that both theNS3/4A gene (SEQ. ID. NO.: 1) and corresponding peptide (SEQ. ID. NO.:2) were immunogenic in vivo.

Mutants of the novel NS3/4A peptide were created. It was discovered thattruncated mutants (e.g., SEQ. ID. NOs.: 12 and 13) and mutants that lacka proteolytic cleavage site (SEQ. ID. NOs.: 3-11), were also immunogenicin vivo. These novel peptides (SEQ. ID. NOs.: 3-13) and fragmentsthereof (e.g., SEQ. ID. NOs.: 16-26) that are any number of consecutiveamino acids between at least 3-50 (e.g., 3, 4, 6, 8, 10, 12, 15, 20, 25,30, 35, 40, 45, or 50 amino acids in length), nucleic acids encodingthese molecules, vectors having said nucleic acids, and cells havingsaid vectors, nucleic acids, or peptides are also embodiments of theinvention.

A codon-optimized nucleic acid encoding NS3/4a was also created and wasfound to be immunogenic. The nucleic acid of SEQ. ID. NO.: 1 wasanalyzed for codon usage and the sequence was compared to the codonsthat are most commonly used in human cells. Because HCV is a humanpathogen, it was unexpected to discover that the virus had not yetevolved to use codons that are most frequently found to encode humanproteins (e.g., optimal human codons). A total of 435 nucleotides werereplaced to generate the codon-optimized synthetic NS3/4A nucleic acid.The NS3/4A peptide encoded by the codon-optimized nucleic acid sequence(SEQ. ID. NO.: 36) was 98% homologous to HCV-1 and contained a total of15 different amino acids.

The codon optimized nucleic acid (MSLF1) (SEQ. ID. NO.: 35) was found tobe more efficiently translated in vitro than the native NS3/4A and thatmice immunized with the MSLF1 containing construct generatedsignificantly more NS3/4A specific antibodies than mice immunized with awild-type NS3/4A containing construct. Further, mice immunized with theMSLF1 containing construct were found to prime NS3-specific CTLs moreeffectively and exhibit better in vivo tumor inhibiting immune responsesthan mice immunized with wild-type NS3/4A containing constructs.

The peptides and nucleic acids described above are useful as immunogens,which can be administered alone or in conjunction with an adjuvant.Preferred embodiments include compositions that comprise one or more ofthe nucleic acids and/or peptides described above with or without anadjuvant. That is, some of the compositions described herein areprepared with or without an adjuvant and comprise, consist, or consistessentially of a NS3/4A peptide (SEQ. ID. NO.: 2 or SEQ. ID. NO.: 36) orfragments thereof that are any number of consecutive amino acids betweenat least 3-50 (e.g., 3, 4, 6, 8, 10, 12, 15, 20, 25, 30, 35, 40, 45, or50 amino acids in length) (e.g., SEQ. ID. NOs.: 14 and 15) or a nucleicacid encoding one or more of these molecules (e.g., SEQ. ID. NO.: 35 ora fragment thereof that is any number of consecutive nucleotides betweenat least 12-2112 (e.g., 12-15, 15-20, 20-30, 30-50, 50-100, 100-200,200-500, 500-1000, 1000-1500, 1500-2079, or 1500-2112 consecutivenucleotides in length). Additional compositions are prepared with orwithout an adjuvant and comprise, consist, or consist essentially of oneor more of the NS3/4A mutant peptides (SEQ. ID. NOs.: 3-13) andfragments thereof that are any number of consecutive amino acids betweenat least 3-50 (e.g., 3, 4, 6, 8, 10, 12, 15, 20, 25, 30, 35, 40, 45, or50 amino acids in length).

It was also discovered that compositions comprising ribavirin and anantigen (e.g., one or more of the previously described HCV peptides ornucleic acids) enhance and/or facilitate an animal's immune response tothe antigen. That is, it was discovered that ribavirin is a veryeffective “adjuvant,” which for the purposes of this disclosure, refersto a material that has the ability to enhance or facilitate an immuneresponse to a particular antigen. The adjuvant activity of ribavirin wasmanifested by a significant increase in immune-mediated protectionagainst the antigen, an increase in the titer of antibody raised to theantigen, and an increase in proliferative T cell responses.

Accordingly, compositions (e.g., vaccines and other medicaments) thatcomprise ribavirin and one or more of the peptides or nucleic acidsdescribed herein are embodiments of the invention. These compositionscan vary according to the amount of ribavirin, the form of ribavirin, aswell as the sequence of the HCV nucleic acid or peptide.

Embodiments of the invention also include methods of making and usingthe compositions above. Some methods involve the making of nucleic acidsencoding NS3/4A, codon-optimized NS3/4A, mutant NS34A, fragments thereofthat are any number of consecutive nucleotides between at least 9-100(e.g., 9, 12, 15, 18, 21, 24, 27, 30, 50, 60, 75, 80, 90, or 100consecutive nucleotides in length), peptides corresponding to saidnucleic acids, constructs comprising said nucleic acids, and cellscontaining said compositions. Preferred methods, however, concern themaking of vaccine compositions or immunogenic preparations thatcomprise, consist, or consist essentially of the newly discovered NS3/4Afragment, codon-optimized NS3/4A, or an NS3/4A mutant (e.g., a truncatedmutant or a mutant lacking a proteolytic cleavage site), or a fragmentthereof or a nucleic acid encoding one or more of these molecules, asdescribed above. Preferred fragments for use with the methods describedherein include SEQ. ID. NOs.: 12-27 and fragments of SEQ. ID. NO.: 35that contain at least 30 consecutive nucleotides. The compositionsdescribed above can be made by providing an adjuvant (e.g., ribavirin),providing an HCV antigen (e.g., a peptide comprising an HCV antigen suchas (SEQ. ID. NOs.: 2-11 or 36) or a fragment thereof such as, SEQ. ID.NOs.: 12-26 or a nucleic acid encoding one or more of said peptides),and mixing said adjuvant and said antigen so as to formulate acomposition that can be used to enhance or facilitate an immune responsein a subject to said antigen.

Methods of enhancing or promoting an immune response in an animal,including humans, to an antigen are also provided. Such methods can bepracticed, for example, by identifying an animal in need of an immuneresponse to HCV and providing said animal a composition comprising oneor more of the nucleic acids or peptides above and an amount of adjuvantthat is effective to enhance or facilitate an immune response to theantigen/epitope. In some embodiments, the antigen and the adjuvant areadministered separately, instead of in a single mixture. Preferably, inthis instance, the adjuvant is administered a short time before or ashort time after administering the antigen. Preferred methods involveproviding the animal in need with ribavirin and NS3/4A (e.g., SEQ. ID.NO.: 2), codon-optimized NS3/4A (e.g., SEQ. ID. NO.: 36), a mutantNS3/4A (e.g., SEQ. ID. NOs.: 3-13), a fragment thereof (e.g., SEQ. ID.NOs.: 14-26) containing any number of consecutive amino acids between atleast 3-50 (e.g., 3, 4, 6, 8, 10, 12, 15, 20, 25, 30, 35, 40, 45, or 50amino acids in length) or a nucleic acid encoding any one or more ofsaid molecules.

Other embodiments concern methods of treating and preventing HCVinfection. By one approach, an immunogen comprising one or more of theHCV nucleic acids or peptides described herein are used to prepare amedicament for the treatment and/or prevention of HCV infection. Byanother approach, an individual in need of a medicament that preventsand/or treats HCV infection is identified and said individual isprovided a medicament comprising ribavirin and an HCV antigen such asNS3/4A (e.g., SEQ. ID. NO.: 2), codon-optimized NS3/4A (e.g., SEQ. ID.NO.: 36), or a mutant NS3/4A (e.g., SEQ. ID. NOs.: 3-13), a fragmentthereof (e.g., SEQ. ID. NOs.: 14-26) containing any number ofconsecutive amino acids between at least 3-50 (e.g., 3, 4, 6, 8, 10, 12,15, 20, 25, 30, 35, 40, 45, or 50 amino acids in length) or a nucleicacid encoding any one or more of these molecules.

The section below discusses the discovery of the novel NS3/4A gene, thecodon-optimized NS3/4A gene, the creation of the NS3/4A mutants, and thecharacterization of the nucleic acids and peptides correspondingthereto.

NS3/4A, NS3/4a Mutants, and Codon-Optimized NS3/4a

A novel nucleic acid and protein corresponding to the NS3/4A domain ofHCV was cloned from a patient infected with HCV (SEQ. ID. NOs.: 1 and2). A Genebank search revealed that the cloned sequence had the greatesthomology to HCV sequences but was only 93% homologous to the closest HCVrelative (accession no AJ 278830). A truncated mutant of the novelNS3/4A peptide and NS3/4A mutants, which lack a proteolytic cleavagesite, (as well as corresponding nucleic acids) were also created.Further, a human codon-optimized NS3/4A nucleic acid and peptide werecreated. It was discovered that these novel peptides and nucleic acidsencoding said peptides were potent immunogens that can be mixed withadjuvants so as to make a composition that induces a recipient toprovide an immune response to HCV. The cloning of the novel NS3/4A geneand the creation of the various NS3/4A mutants and codon optimizedNS3/4A gene are described in the following example.

Example 1

The NS3/4A sequence was amplified from the serum of an HCV-infectedpatient (HCV genotype 1a) using the Polymerase Chain Reaction (PCR).Total RNA was extracted from serum, and cDNA synthesis and PCR wereperformed according to standard protocols (Chen M et al., J. Med. Virol.43:223-226 (1995)). The cDNA synthesis was initiated using the antisenseprimer “NS4KR” (5′-CCG TCT AGA TCA GCA CTC TTC CAT TTC ATC-3′ (SEQ. ID.NO.: 28)). From this cDNA, a 2079 base pair DNA fragment of HCV,corresponding to amino acids 1007 to 1711, which encompasses the NS3 andNS4A genes, was amplified. A high fidelity polymerase (Expand HighFidelity PCR, Boehringer-Mannheim, Mannheim, Germany) was used with the“NS3KF” primer (5′-CCT GAA TTC ATG GCG CCT ATC ACG GCC TAT-3′ (SEQ. ID.NO.: 29) and the NS4KR primer. The NS3KF primer contained a EcoRIrestriction enzyme cleavage site and a start codon and the primer NS4KRcontained a XbaI restriction enzyme cleavage site and a stop codon.

The amplified fragment was then sequenced (SEQ. ID. NO.: 1). Sequencecomparison analysis revealed that the gene fragment was amplified from aviral strain of genotype 1a. A computerized BLAST search against theGenbank database using the NCBI website revealed that the closest HCVhomologue was 93% identical in nucleotide sequence.

The amplified DNA fragment was then digested with EcoRI and XbaI, andwas inserted into a pcDNA3.1/His plasmid (Invitrogen) digested with thesame enzymes. The NS3/4A-pcDNA3.1 plasmid was then digested with EcoRIand Xba I and the insert was purified using the QiaQuick kit (Qiagen,Hamburg, Germany) and was ligated to a EcoRI/Xba I digested pVAX vector(Invitrogen) so as to generate the NS3/4A-pVAX plasmid.

The rNS3 truncated mutant was obtained by deleting NS4A sequence fromthe NS3/4A DNA. Accordingly, the NS3 gene sequence of NS3/4A-pVAX wasPCR amplified using the primers NS3KF and 3′NotI (5′-CCA CGC GGC CGC GACGAC CTA CAG-3′ (SEQ. ID. NO.: 30)) containing EcoRI and Not Irestriction sites, respectively. The NS3 fragment (1850 bp) was thenligated to a EcoRI and Not I digested pVAX plasmid to generate theNS3-pVAX vector. Plasmids were grown in BL21 E. coli cells. The plasmidswere sequenced and were verified by restriction cleavage and the resultswere as to be expected based on the original sequence.

Table 1 describes the sequence of the proteolytic cleavage site ofNS3/4A, referred to as the breakpoint between NS3 and NS4A. Thiswild-type breakpoint sequence was mutated in many different ways so asto generate several different NS3/4A breakpoint mutants. Table 1 alsoidentifies these mutant breakpoint sequences. The fragments listed inTABLE 1 are preferred immunogens that can be incorporated with orwithout an adjuvant (e.g., ribavirin) into a composition foradministration to an animal so as to induce an immune response in saidanimal to HCV.

To change the proteolytic cleavage site between NS3 and NS4A, theNS3/4A-pVAX plasmid was mutagenized using the QUICKCHANGE™ mutagenesiskit (Stratagene), following the manufacturer's recommendations. Togenerate the “TPT” mutation, for example, the plasmid was amplifiedusing the primers 5″-CTGGAGGTCGTCACGCCTACCTGGGTGCTCGTT-3′ (SEQ. ID. NO.:31) and 5″-ACCGAGCACCCAGGTAGGCGTGACGACCTCCAG-3′ (SEQ. ID. NO.: 32)resulting in NS3/4A-TPT-pVAX. To generate the “RGT” mutation, forexample, the plasmid was amplified using the primers5′-CTGGAGGTCGTCCGCGGTACCTGGGTGCTCGTT-3′ (SEQ. ID. NO.: 33) and5′-ACCGAGCACCCAGGTACC-GCGGACGACCTCCAG-3′ (SEQ. ID. NO.: 34) resulting inNS3/4A-RGT-pVAX. All mutagenized constructs were sequenced to verifythat the mutations had been correctly made. Plasmids were grown incompetent BL21 E. coli.

The sequence of the previously isolated and sequenced unique NS3/4A gene(SEQ. ID. NO.: 1) was analyzed for codon usage with respect to the mostcommonly used codons in human cells. A total of 435 nucleotides werereplaced to optimize codon usage for human cells. The sequence was sentto Retrogen Inc. (6645 Nancy Ridge Drive, San Diego, Calif. 92121) andthey were provided with instructions to generate a full-length syntheticcodon optimized NS3/4A gene. The codon optimized NS3/4A gene had asequence homology of 79% within the region between nucleotide positions3417-5475 of the HCV-1 reference strain. A total of 433 nucleotidesdiffered. On an amino acid level, the homology with the HCV-1 strain was98% and a total of 15 amino acids differed.

The full length codon optimized 2.1 kb DNA fragment of the HCVcorresponding to the amino acids 1007 to 1711 encompassing the NS3 andNS4A NS3/NS4A gene fragment was amplified by the polymerase chainreaction (PCR) using high fidelity polymerase (Expand High Fidelity PCR,Boehringer-Mannheim, Mannheim, Germany). The amplicon was then insertedinto a Bam HI and Xba I digested pVAX vector (Invitrogen, San Diego),which generated the MSLF1-pVAX plasmid. All expression constructs weresequenced. Plasmids were grown in competent BL21 E. Coli. The plasmidDNA used for in vivo injection was purified using Qiagen DNApurification columns, according to the manufacturers instructions(Qiagen GmbH, Hilden, FRG). The concentration of the resulting plasmidDNA was determined spectrophotometrically (Dynaquant, Pharmacia Biotech,Uppsala, Sweden) and the purified DNA was dissolved in sterile phosphatebuffer saline (PBS) at concentrations of 1 mg/ml.

TABLE 1 Plasmid Deduced amino acid sequence *NS3/4A-pVAXTKYMTCMSADLEVVTSTWVLVGGVL (SEQ. ID. NO.: 14) NS3/4A-TGT-pVAXTKYMTCMSADLEVVTGTWVLVGGVL (SEQ. ID. NO.: 16) NS3/4A-RGT-pVAXTKYMTCMSADLEVVRGTWVLVGGVL (SEQ. ID. NO.: 17) NS3/4A-TPT-pVAXTKYMTCMSADLEVVTPTWVLVGGVL (SEQ. ID. NO.: 18) NS3/4A-RPT-pVAXTKYMTCMSADLEVVRPTWVLVGGVL (SEQ. ID. NO.: 19) NS3/4A-RPA-pVAXTKYMTCMSADLEVVRPAWVLVGGVL (SEQ. ID. NO.: 20) NS3/4A-CST-pVAXTKYMTCMSADLEVVCSTWVLVGGVL (SEQ. ID. NO.: 21) NS3/4A-CCST-pVAXTKYMTCMSADLEVCCSTWVLVGGVL (SEQ. ID. NO.: 22) NS3/4A-SSST-pVAXTKYMTCMSADLEVSSSTWVLVGGVL (SEQ. ID. NO.: 23) NS3/4A-SSSSCST-pVAXTKYMTCMSADSSSSCSTWVLVGGVL (SEQ. ID. NO.: 24) NS3A/4A-VVVVTST-pVAXTKYMTCMSADVVVVTSTWVLVGGVL (SEQ. ID. NO.: 25) NS5-pVAX ASEDVVCCSMSYTWTG(SEQ. ID. NO.: 27) NS5A/B-pVAX SSEDVVCCSMWVLVGGVL (SEQ. ID. NO.: 26)*The wild type sequence for the NS3/4A fragment is NS3/4A-pVAX. TheNS3/4A breakpoint is identified by underline, wherein the P1 positioncorresponds to the first Thr (T) and the P1′ position corresponds to thenext following amino acid the NS3/4A-pVAX sequence. In the wild typeNS3/4A sequence the NS3 protease cleaves between the P1 and P1′positions.

Several nucleic acid embodiments include nucleotides encoding the HCVpeptides described herein (SEQ. ID. NOs.: 2-11 or SEQ. ID. NO.: 36) or afragment thereof (e.g., SEQ. ID. NOs.: 14 and 15) containing any numberof consecutive amino acids between at least 3-50 (e.g., 3, 4, 6, 8, 10,12, 15, 20, 25, 30, 35, 40, 45, or 50 amino acids in length). Someembodiments for example, include genomic DNA, RNA, and cDNA encodingthese HCV peptides. The HCV nucleotide embodiments not only include theDNA sequences shown in the sequence listing (e.g., SEQ. ID. NO.: 1 orSEQ. ID. NO.: 35) but also include nucleotide sequences encoding theamino acid sequences shown in the sequence listing (e.g., SEQ. ID. NOs.:2-11 or SEQ. ID. NO.: 36) and any nucleotide sequence that hybridizes tothe DNA sequences shown in the sequence listing under stringentconditions (e.g., hybridization to filter-bound DNA in 0.5 M NaHPO₄,7.0% sodium dodecyl sulfate (SDS), 1 mM EDTA at 50° C.) and washing in0.2×SSC/0.2% SDS at 50° C. and any nucleotide sequence that hybridizesto the DNA sequences that encode an amino acid sequence provided in thesequence listing (SEQ. ID. NOs.: 2-11 or SEQ. ID. NO.: 36) under lessstringent conditions (e.g., hybridization in 0.5 M NaHPO₄, 7.0% sodiumdodecyl sulfate (SDS), 1 mM EDTA at 37° C. and washing in 0.2×SSC/0.2%SDS at 37° C.).

The nucleic acid embodiments of the invention also include fragments,modifications, derivatives, and variants of the sequences describedabove. Desired embodiments, for example, include nucleic acids having atleast 25 consecutive bases of one of the novel HCV sequences or asequence complementary thereto and preferred fragments include at least25 consecutive bases of a nucleic acid encoding the NS3/4A molecule ofSEQ. ID. NO.: 2 or SEQ. ID. NO.: 36 or a mutant NS3/4A molecule of SEQ.ID. NOs.: 3-13 or a sequence complementary thereto.

In this regard, the nucleic acid embodiments described herein can haveany number of consecutive nucleotides between about 12 to approximately2112 consecutive nucleotides of SEQ. ID. NO.: 1 or SEQ. ID. NO.: 35.Some DNA fragments, for example, include nucleic acids having at least12-15, 15-20, 20-30, 30-50, 50-100, 100-200, 200-500, 500-1000,1000-1500, 1500-2079, or 1500-2112 consecutive nucleotides of SEQ. ID.NO.: 1 or SEQ. ID. NO.: 35 or a complement thereof. These nucleic acidembodiments can also be altered by substitution, addition, or deletionso long as the alteration does not significantly affect the structure orfunction (e.g., ability to serve as an immunogen) of the HCV nucleicacid. Due to the degeneracy of nucleotide coding sequences, for example,other DNA sequences that encode substantially the same HCV amino acidsequence as depicted in SEQ. ID. NOs.: 2-13 or SEQ. ID. NO.: 36 can beused in some embodiments. These include, but are not limited to, nucleicacid sequences encoding all or portions of HCV peptides (SEQ. ID. NOs.:2-13) or nucleic acids that complement all or part of this sequence thathave been altered by the substitution of different codons that encode afunctionally equivalent amino acid residue within the sequence, thusproducing a silent change, or a functionally non-equivalent amino acidresidue within the sequence, thus producing a detectable change.Accordingly, the nucleic acid embodiments of the invention are said tobe comprising, consisting of, or consisting essentially of nucleic acidsencoding any one of SEQ. ID. NOs.: 2-27 or SEQ. ID. NO.: 36 in light ofthe modifications above.

By using the nucleic acid sequences described above, probes thatcomplement these molecules can be designed and manufactured byoligonucleotide synthesis. Desirable probes comprise a nucleic acidsequence of (SEQ. ID. NO.: 1) that is unique to this HCV isolate. Theseprobes can be used to screen cDNA from patients so as to isolate naturalsources of HCV, some of which may be novel HCV sequences in themselves.Screening can be by filter hybridization or by PCR, for example. Byfilter hybridization, the labeled probe preferably contains at least15-30 base pairs of the nucleic acid sequence of (SEQ. ID. NO.: 1) thatis unique to this NS3/4A peptide. The hybridization washing conditionsused are preferably of a medium to high stringency. The hybridizationcan be performed in 0.5M NaHPO₄, 7.0% sodium dodecyl sulfate (SDS), 1 mMEDTA at 42° C. overnight and washing can be performed in 0.2×SSC/0.2%SDS at 42° C. For guidance regarding such conditions see, for example,Sambrook et al., 1989, Molecular Cloning, A Laboratory Manual, ColdSprings Harbor Press, N.Y.; and Ausubel et al., 1989, Current Protocolsin Molecular Biology, Green Publishing Associates and WileyInterscience, N.Y.

HCV nucleic acids can also be isolated from patients infected with HCVusing the nucleic acids described herein. (See also Example 1).Accordingly, RNA obtained from a patient infected with HCV is reversetranscribed and the resultant cDNA is amplified using PCR or anotheramplification technique. The primers are preferably obtained from theNS3/4A sequence (SEQ. ID. NO.: 1).

For a review of PCR technology, see Molecular Cloning to GeneticEngineering White, B. A. Ed. in Methods in Molecular Biology 67: HumanaPress, Totowa (1997) and the publication entitled “PCR Methods andApplications” (1991, Cold Spring Harbor Laboratory Press). Foramplification of mRNAs, it is within the scope of the invention toreverse transcribe mRNA into cDNA followed by PCR (RT-PCR); or, to use asingle enzyme for both steps as described in U.S. Pat. No. 5,322,770.Another technique involves the use of Reverse Transcriptase AsymmetricGap Ligase Chain Reaction (RT-AGLCR), as described by Marshall R. L. etal. (PCR Methods and Applications 4:80-84, 1994).

Briefly, RNA is isolated, following standard procedures. A reversetranscription reaction is performed on the RNA using an oligonucleotideprimer specific for the most 5′ end of the amplified fragment as aprimer of first strand synthesis. The resulting RNA/DNA hybrid is then“tailed” with guanines using a standard terminal transferase reaction.The hybrid is then digested with RNAse H, and second strand synthesis isprimed with a poly-C primer. Thus, cDNA sequences upstream of theamplified fragment are easily isolated. For a review of cloningstrategies which can be used, see e.g., Sambrook et al., 1989, supra.

In each of these amplification procedures, primers on either side of thesequence to be amplified are added to a suitably prepared nucleic acidsample along with dNTPs and a thermostable polymerase, such as Taqpolymerase, Pfu polymerase, or Vent polymerase. The nucleic acid in thesample is denatured and the primers are specifically hybridized tocomplementary nucleic acid sequences in the sample. The hybridizedprimers are then extended. Thereafter, another cycle of denaturation,hybridization, and extension is initiated. The cycles are repeatedmultiple times to produce an amplified fragment containing the nucleicacid sequence between the primer sites. PCR has further been describedin several patents including U.S. Pat. Nos. 4,683,195, 4,683,202 and4,965,188, all of which are expressly incorporated by reference in theirentireties.

The primers are selected to be substantially complementary to a portionof the nucleic acid sequence of (SEQ. ID. NO.: 1) that is unique to thisNS3/4A molecule, thereby allowing the sequences between the primers tobe amplified. Preferably, primers can be any number between at least16-20, 20-25, or 25-30 nucleotides in length. The formation of stablehybrids depends on the melting temperature (Tm) of the DNA. The Tmdepends on the length of the primer, the ionic strength of the solutionand the G+C content. The higher the G+C content of the primer, thehigher is the melting temperature because G:C pairs are held by three Hbonds whereas A:T pairs have only two. The G+C content of theamplification primers described herein preferably range between 10% and75%, more preferably between 35% and 60%, and most preferably between40% and 55%. The appropriate length for primers under a particular setof assay conditions can be empirically determined by one of skill in theart.

The spacing of the primers relates to the length of the segment to beamplified. In the context of the embodiments described herein, amplifiedsegments carrying nucleic acid sequence encoding HCV peptides can rangein size from at least about 25 bp to the entire length of the HCVgenome. Amplification fragments from 25-1000 bp are typical, fragmentsfrom 50-1000 bp are preferred and fragments from 100-600 bp are highlypreferred. It will be appreciated that amplification primers can be ofany sequence that allows for specific amplification of the NS3/4A regionand can, for example, include modifications such as restriction sites tofacilitate cloning.

The PCR product can be subcloned and sequenced to ensure that theamplified sequences represent the sequences of an HCV peptide. The PCRfragment can then be used to isolate a full length cDNA clone by avariety of methods. For example, the amplified fragment can be labeledand used to screen a cDNA library, such as a bacteriophage cDNA library.Alternatively, the labeled fragment can be used to isolate genomicclones via the screening of a genomic library. Additionally, anexpression library can be constructed utilizing cDNA synthesized from,for example, RNA isolated from an infected patient. In this manner, HCVgene products can be isolated using standard antibody screeningtechniques in conjunction with antibodies raised against the HCV geneproduct. (For screening techniques, see, for example, Harlow, E. andLane, eds., 1988, Antibodies: A Laboratory Manual, Cold Spring HarborPress, Cold Spring Harbor).

Embodiments of the invention also include (a) DNA vectors that containany of the foregoing nucleic acid sequence and/or their complements(i.e., antisense); (b) DNA expression vectors that contain any of theforegoing nucleic acid sequences operatively associated with aregulatory element that directs the expression of the nucleic acid; and(c) genetically engineered host cells that contain any of the foregoingnucleic acid sequences operatively associated with a regulatory elementthat directs the expression of the coding sequences in the host cell.These recombinant constructs are capable of replicating autonomously ina host cell. Alternatively, the recombinant constructs can becomeintegrated into the chromosomal DNA of a host cell. Such recombinantpolynucleotides typically comprise an HCV genomic or cDNA polynucleotideof semi-synthetic or synthetic origin by virtue of human manipulation.Therefore, recombinant nucleic acids comprising these sequences andcomplements thereof that are not naturally occurring are provided.

Although nucleic acids encoding an HCV peptide or nucleic acids havingsequences that complement an HCV gene as they appear in nature can beemployed, they will often be altered, e.g., by deletion, substitution,or insertion, and can be accompanied by sequence not present in humans.As used herein, regulatory elements include, but are not limited to,inducible and non-inducible promoters, enhancers, operators and otherelements known to those skilled in the art that drive and regulateexpression. Such regulatory elements include, but are not limited to,the cytomegalovirus hCMV immediate early gene, the early or latepromoters of SV40 adenovirus, the lac system, the trp system, the TACsystem, the TRC system, the major operator and promoter regions of phageA, the control regions of fd coat protein, the promoter for3-phosphoglycerate kinase, the promoters of acid phosphatase, and thepromoters of the yeast-mating factors.

In addition, recombinant HCV peptide-encoding nucleic acid sequences andtheir complementary sequences can be engineered so as to modify theirprocessing or expression. For example, and not by way of limitation, theHCV nucleic acids described herein can be combined with a promotersequence and/or ribosome binding site, or a signal sequence can beinserted upstream of HCV peptide-encoding sequences so as to permitsecretion of the peptide and thereby facilitate harvesting orbioavailability. Additionally, a given HCV nucleic acid can be mutatedin vitro or in vivo, to create and/or destroy translation, initiation,and/or termination sequences, or to create variations in coding regionsand/or form new restriction sites or destroy preexisting ones, or tofacilitate further in vitro modification. (See Example 1). Any techniquefor mutagenesis known in the art can be used, including but not limitedto, in vitro site-directed mutagenesis. (Hutchinson et al., J. Biol.Chem., 253:6551 (1978)). The nucleic acids encoding the HCV peptides,described above, can be manipulated using conventional techniques inmolecular biology so as to create recombinant constructs that expressthe HCV peptides.

Further, nucleic acids encoding other proteins or domains of otherproteins can be joined to nucleic acids encoding an HCV peptide so as tocreate a fusion protein. Nucleotides encoding fusion proteins caninclude, but are not limited to, a full length NS3/4A sequence (SEQ. ID.NO.: 2 or SEQ. ID. NO.: 36), mutant NS3/4A sequences (e.g., SEQ. ID.NOs.: 3-11) or a peptide fragment of an NS3/4A sequence fused to anunrelated protein or peptide, such as for example, polyhistidine,hemagglutinin, an enzyme, fluorescent protein, or luminescent protein,as discussed below.

It was discovered that the construct “NS3/4A-pVAX” was significantlymore immunogenic in vivo than the construct “NS3-pVAX”. Surprisingly, itwas also discovered that the codon-optimized NS3/4A containing construct(“MSLF1-pVAX”) was more immunogenic in vivo than NS3/4A pVAX. Theexample below describes these experiments.

Example 2

To determine whether a humoral immune response was elicited by theNS3-pVAX and NS3/4A-pVAX vectors, the expression constructs described inExample 1 were purified using the Qiagen DNA purification system,according to the manufacturer's instructions and the purified DNAvectors were used to immunize groups of four to ten Balb/c mice. Theplasmids were injected directly into regenerating tibialis anterior (TA)muscles as previously described (Davis et al., Human Gene Therapy4(6):733 (1993)). In brief, mice were injected intramuscularly with 50μl/TA of 0.01 mM cardiotoxin (Latoxan, Rosans, France) in 0.9% sterileNaCl. Five days later, each TA muscle was injected with 50 μl PBScontaining either rNS3 or DNA.

Inbred mouse strains C57/BL6 (H-2b), Balb/C(H-2d), and CBA (H-2k) wereobtained from the breeding facility at Möllegard Denmark, Charles RiverUppsala, Sweden, or B&K Sollentuna Sweden. All mice were female and wereused at 4-8 weeks of age. For monitoring of humoral responses, all micereceived a booster injection of 50 μl/TA of plasmid DNA every fourthweek. In addition, some mice were given recombinant NS3 (rNS3) protein,which was purified, as described herein. The mice receiving rNS3 wereimmunized no more than twice. All mice were bled twice a month.

Enzyme immunosorbent assays (EIAs) were used to detect the presence ofmurine NS3-specific antibodies. These assays were performed essentiallyas described (Chen et al., Hepatology 28(1): 219 (1998), hereinexpressly incorporated by reference in its entirety). Briefly, rNS3 waspassively adsorbed overnight at 4° C. to 96-well microtiter plates(Nunc, Copenhagen, Denmark) at 1 μg/ml in 50 mM sodium carbonate buffer(pH 9.6). The plates were then blocked by incubation with dilutionbuffer containing PBS, 2% goat serum, and 1% bovine serum albumin forone hour at 37° C. Serial dilutions of mouse sera starting at 1:60 werethen incubated on the plates for one hour. Bound murine serum antibodieswere detected by an alkaline phosphatase conjugated goat anti-mouse IgG(Sigma Cell Products, Saint Louis, Mo.) followed by addition of thesubstrate pNPP (1 tablet/5 ml of 1M Diethanol amine buffer with 0.5 mMMgCl₂). The reaction was stopped by addition of 1M NaOH and absorbencywas read at 405 nm.

After four weeks, four out of five mice immunized with NS3/4A-pVAX haddeveloped NS3 antibodies, whereas one out of five immunized withNS3-pVAX had developed antibodies (FIG. 1). After six weeks, four out offive mice immunized with NS3/4A-pVAX had developed high levels (>10⁴) ofNS3 antibodies (mean levels 10800±4830) and one had a titer of 2160.Although all mice immunized with NS3-pVAX developed NS3 antibodies, noneof them developed levels as high as that produced by the NS3/4A-pVAXconstruct (mean levels 1800±805). The antibody levels elicited by theNS3/4A fusion construct were significantly higher than those induced byNS3-pVAX at six weeks (mean ranks 7.6 v.s 3.4, p<0.05, Mann-Whitney ranksum test, and p<0.01, Students t-test). Thus, immunization with eitherNS3-pVAX or NS3/4A-pVAX resulted in the production of NS3-specificantibodies, but the NS3/4A containing construct was a more potentimmunogen.

A similar experiment was conducted to compare the immunogenicity of theNS3/4A-pVAX and MSLF1-pVAX constructs. To better resemble a futurevaccination schedule in humans, however, the plasmids were delivered togroups of ten mice using a gene gun. In brief, plasmid DNA was linked togold particles according to protocols supplied by the manufacturer(Bio-Rad Laboratories, Hercules, Calif.). Prior to immunization, theinjection area was shaved and the immunization was performed accordingto the manufacturer's protocol. Each injection dose contained 4 μg ofplasmid DNA. Immunizations were performed on weeks 0, 4, and 8.

The MSLF 1 gene was found to be more immunogenic than the native NS3/4Agene since NS3-specific antibodies were significantly higher in miceimmunized with the MSLF1-pVAX construct at two weeks after the secondand third immunization (TABLE 2). These results confirmed thatMSLF1-pVAX was a more potent B cell immunogen than NS3/4A-pVAX.

TABLE 2 No. of Mean Immunogen Week injections NS3 titre SD Mann-WhitneyNS3/4A 2 1 0 0 NS MSLF1 2 1 0 0 NS3/4A 6 2 0 0 p < 0.0002 MSLF1 6 2 24843800 NS3/4A 10 3 60 0 p < 0.0001 MSLF1 10 3 4140 4682

The example below describes experiments that were performed to determineif mutant NS3/4A peptides, which lack a proteolytic cleavage site, couldelicit an immune response to NS3.

Example 3

To test if the enhanced immunogenicity of NS3/4A could be solelyattributed to the presence of NS4A, or if the NS3/4A fusion protein inaddition had to be cleaved at the NS3/4A junction, another set ofexperiments were performed. In a first experiment, the immunogenicity ofthe NS3-pVAX, NS3/4A-pVAX, and mutant NS3/4A constructs were compared inBalb/c mice. Mice were immunized on week 0 as described above, and,after two weeks, all mice were bled and the presence of antibodies toNS3 at a serum dilution of 1:60 was determined (TABLE 3). Mice were bledagain on week 4. As shown in TABLE 3, all the constructs induced animmune response; the mutant constructs, for example, the NS3/4A-TGT-pVAXvector was comparable to the NS3-pVAX vector (4/10 vs. 0/10; NS,Fisher's exact test). The NS3/4A-pVAX vector, however, was more potentthan the mutant constructs.

TABLE 3 No. of antibody responders to the respective immunogen after one100 μg i.m immunization Weeks from 1^(st) wild-type mutant exampleimmunization NS3-pVAX NS3/4A-pVAX NS3/4A-TGT-pVAX 2 0/10 17/20  4/10 40/10 20/20 10/10 (<60) (2415 ± 3715) (390 ± 639) 55% > 10³ 50% > 10²10% > 10⁴ 10% > 10³

During the chronic phase of infection, HCV replicates in hepatocytes,and spreads within the liver. A major factor in combating chronic andpersistent viral infections is the cell-mediated immune defense system.CD4+ and CD8+ lymphocytes infiltrate the liver during the chronic phaseof HCV infection, but they are incapable of clearing the virus orpreventing liver damage. In addition, persistent HCV infection isassociated with the onset of hepatocellular carcinoma (HCC). Theexamples below describe experiments that were performed to determinewhether the NS3, NS3/4A, and MSLF1 constructs were capable of elicitinga T-cell mediated immune response against NS3.

Example 4

To study whether the constructs described above were capable ofeliciting a cell-mediated response against NS3, an in vivo tumor growthassay was performed. To this end, an SP2/0 tumor cell line (SP2/0-Ag14myeloma cell line (H-2^(d))) stably transfected with the NS3/4A gene wasmade. The SP2/0 cells were maintained in DMEM medium supplemented with10% fetal calf serum (FCS; Sigma Chemicals, St. Louis, Mo.), 2 mML-Glutamine, 10 mM HEPES, 100 U/ml Penicillin and 100 μg/mlStreptomycin, 1 mM non-essential amino acids, 50 μM β-mercaptoethanol, 1mM sodium pyruvate (GIBCO-BRL, Gaithesburgh, Md.). The pcDNA3.1 plasmidcontaining the NS3/4A gene was linearized by BglII digestion. A total of5 μg linearized plasmid DNA was mixed with 60 μg transfection reagent(Superfect, Qiagen, Germany) and the mixture was added to a 50%confluent layer of SP2/0 cells in a 35 mm dish. The transfectionprocedure was performed according to manufacturer's protocol.

Transfected cells were cloned by limiting dilution and selected byaddition of 800 μg geneticin (G418)/ml complete DMEM medium after 14days. A stable NS3/4A-expressing SP2/0 clone was identified using PCRand RTPCR and/or a capture EIA using a monoclonal antibody to NS3. AllEIAs for the detection of murine NS3 antibodies were essentiallyperformed as follows. In brief, rNS3 (recombinant NS3 protein producedin E. Coli, dialyzed overnight against PBS, and sterile filtered) waspassively adsorbed overnight at 4° C. to 96-well microtiter plates(Nunc, Copenhagen, Denmark) at 1 μg/ml in 50 mM sodium carbonate buffer(pH 9.6). The plates were then blocked by incubation with dilutionbuffer containing PBS, 2% goat serum, and 1% bovine serum albumin forone hour at +37° C. Serial dilutions of mouse sera starting at 1:60 werethen incubated on the plates for one hour. Bound murine serum antibodieswere detected by an alkaline phosphatase conjugated goat anti-mouse IgG(Sigma cellproducts, Saint Louis, Mo. USA) followed by addition of thesubstrate pNPP (1 tablet/5 ml of 1M Diethanolamin buffer with 0.5 mMMgCl2). The reaction was stopped by addition of 1M NaOH. Absorbance wasthen read at 405 nm.

The in vivo growth kinetics of the SP2/0 and the NS3/4A-SP2/0 cell lineswere then evaluated in Balb/c mice. Mice were injected subcutaneouslywith 2×10⁶ tumor cells in the right flank. Each day the size of thetumor was determined through the skin. The growth kinetics of the twocell lines was comparable. The mean tumor sizes did not differ betweenthe two cell lines at any time point, for example. (See TABLE 4).

TABLE 4 Mouse Tumor Maximum in vivo tumor size at indicated time pointID cell line 5 6 7 8 11 12 13 14 15 1 SP2/0 1.6 2.5 4.5 6.0 10.0 10.511.0 12.0 12.0 2 SP2/0 1.0 1.0 2.0 3.0 7.5 7.5 8.0 11.5 11.5 3 SP2/0 2.05.0 7.5 8.0 11.0 11.5 12.0 12.0 13.0 4 SP2/0 4.0 7.0 8.0 10.0 13.0 15.016.5 16.5 17.0 5 SP2/0 1.0 1.0 3.0 4.0 5.0 6.0 6.0 6.0 7.0 Group mean1.92 3.3 5.0 6.2 9.3 10.1 10.7 11.6 12.1 6 NS3/4A-SP2/0 1.0 2.0 3.0 3.54.0 5.5 6.0 7.0 8.0 7 NS3/4A-SP2/0 2.0 2.5 3.0 5.0 7.0 9.0 9.5 9.5 11.08 NS3/4A-SP2/0 1.0 2.0 3.5 3.5 9.5 11.0 12.0 14.0 14.0 9 NS3/4A-SP2/01.0 1.0 2.0 6.0 11.5 13.0 14.5 16.0 18.0 10 NS3/4A-SP2/0 3.5 6.0 7.010.5 15.0 15.0 15.0 15.5 20.0 Group mean 1.7 2.7 3.7 5.7 9.4 10.7 11.412.4 14.2 p-value of student's 0.7736 0.6918 0.4027 0.7903 0.9670 0.79860.7927 0.7508 0.4623 t-test comparison between group means

The example below describes experiments that were performed to determinewhether mice immunized with the NS3/4A constructs had developed a T-cellresponse against NS3.

Example 5

To examine whether a T-cell response was elicited by the NS3/4Aimmunization, the capacity of an immunized mouse's immune defense systemto attack the NS3-expressing tumor cell line was assayed. The protocolfor testing for in vivo inhibition of tumor growth of the SP2/0 myelomacell line in Balb/c mice has been described in detail previously (Enckeet al., J. Immunol. 161:4917 (1998), herein expressly incorporated byreference in its entirety). Inhibition of tumor growth in this model isdependent on the priming of cytotoxic T lymphocytes (CTLs). In a firstset of experiments, groups of ten mice were immunized i.m. five timeswith one month intervals with either 100 μg NS3-pVAX or 100 μgNS3/4A-pVAX. Two weeks after the last immunization 2×10⁶ SP2/0 orNS3/4A-SP2/0 cells were injected into the right flank of each mouse. Twoweeks later the mice were sacrificed and the maximum tumor sizes weremeasured. There was no difference between the mean SP2/0 andNS3/4A-SP2/0 tumor sizes in the NS3-pVAX immunized mice. (See TABLE 5).

TABLE 5 Maximum Mouse Dose Tumor Tumor tumor size ID Immunogen (μg) cellline growth (mm) 1 NS3-pVAX 100 SP2/0 Yes 5 2 NS3-pVAX 100 SP2/0 Yes 153 NS3-pVAX 100 SP2/0 No — 4 NS3-pVAX 100 SP2/0 Yes 6 5 NS3-pVAX 100SP2/0 Yes 13 Group total 4/5 9.75 ± 4.992 6 NS3-pVAX 100 NS3/4A-SP2/0Yes 9 7 NS3-pVAX 100 NS3/4A-SP2/0 Yes 8 8 NS3-pVAX 100 NS3/4A-SP2/0 Yes7 9 NS3-pVAX 100 NS3/4A-SP2/0 No — 10 NS3-pVAX 100 NS3/4A-SP2/0 No — 3/58.00 ± 1.00  Note: Statistical analysis (StatView): Student's t-test onmaximum tumor size. P-values < 0.05 are considered significant.

Unpaired t-test for Max diam Grouping Variable: Column 1 HypothesizedDifference = 0 Row exclusion: NS3DNA-Tumor-001213 Mean Diff. DF t-ValueP-Value NS3-sp2, NS3-spNS3 1.750 5 0.58 0.584

Group Info for Max diam Grouping Variable: Column 1 Row exclusion:NS3DNA-Tumor-001213 Count Mean Variance Std. Dev. Std. Err NS3-sp2 49.750 24.917 4.992 2.496 NS3-spNS3 3 8.000 1.000 1.000 0.57

To analyze whether administration of different NS3 containingcompositions affected the elicitation of a cell-mediated immuneresponse, mice were immunized with PBS, rNS3, a control DNA, or theNS3/4A construct, and tumor sizes were determined, as described above.The NS3/4A construct was able to elicit a T-cell response sufficient tocause a statistically significant reduction in tumor size (See TABLE 6).

TABLE 6 Maximum Mouse Dose Tumor Tumor tumor size ID Immunogen (μg) cellline Anti-NS3 growth (mm) 1 NS3-pVAX 10 NS3/4A-SP2/0 <60 + 12.0 2NS3-pVAX 10 NS3/4A-SP2/0 <60 + 20.0 3 NS3-pVAX 10 NS3/4A-SP2/0 60 + 18.04 NS3-pVAX 10 NS3/4A-SP2/0 <60 + 13.0 5 NS3-pVAX 10 NS3/4A-SP2/0 <60 +17.0 Group mean 60 5/5  16.0 ± 3.391 6 NS3-pVAX 100 NS3/4A-SP2/0 2160 +10.0 7 NS3-pVAX 100 NS3/4A-SP2/0 <60 − — 8 NS3-pVAX 100 NS3/4A-SP2/0 <60− — 9 NS3-pVAX 100 NS3/4A-SP2/0 360 − — 10 NS3-pVAX 100 NS3/4A-SP2/0<60 + 12.5 Group mean 1260 2/5 11.25 ± 1.768 11 NS3/4A-pVAX 10NS3/4A-SP2/0 <60 + 10.0 12 NS3/4A-pVAX 10 NS3/4A-SP2/0 <60 − — 13NS3/4A-pVAX 10 NS3/4A-SP2/0 <60 − — 14 NS3/4A-pVAX 10 NS3/4A-SP2/0 <60 +13.0 15 NS3/4A-pVAX 10 NS3/4A-SP2/0 <60 + 13.5 Group mean <60 3/5 12.167± 1.893  16 NS3/4A-pVAX 100 NS3/4A-SP2/0 60 + 10.0 17 NS3/4A-pVAX 100NS3/4A-SP2/0 360 − — 18 NS3/4A-pVAX 100 NS3/4A-SP2/0 2160 + 8.0 19NS3/4A-pVAX 100 NS3/4A-SP2/0 2160 + 12.0 20 NS3/4A-pVAX 100 NS3/4A-SP2/02160 + 7.0 Group mean 1380 4/5  9.25 ± 2.217 36 p17-pcDNA3 100NS3/4A-SP2/0 <60 + 20.0 37 p17-pcDNA3 100 NS3/4A-SP2/0 <60 + 7.0 38p17-pcDNA3 100 NS3/4A-SP2/0 <60 + 11.0 39 p17-pcDNA3 100 NS3/4A-SP2/0<60 + 15.0 40 p17-pcDNA3 100 NS3/4A-SP2/0 <60 + 18.0 Group mean <60 5/514.20 ± 5.263 41 rNS3/CFA 20 NS3/4A-SP2/0 >466560 + 13.0 42 rNS3/CFA 20NS3/4A-SP2/0 >466560 − — 43 rNS3/CFA 20 NS3/4A-SP2/0 >466560 + 3.5 44rNS3/CFA 20 NS3/4A-SP2/0 >466560 + 22.0 45 rNS3/CFA 20NS3/4A-SP2/0 >466560 + 17.0 Group mean 466560 4/5 17.333 ± 4.509  46 PBS— NS3/4A-SP2/0 <60 + 10.0 47 PBS — NS3/4A-SP2/0 <60 + 16.5 48 PBS —NS3/4A-SP2/0 60 + 15.0 49 PBS — NS3/4A-SP2/0 <60 + 21.0 50 PBS —NS3/4A-SP2/0 <60 + 15.0 51 PBS — NS3/4A-SP2/0 <60 − — Group mean 60 5/615.50 ± 3.937 Note: Statistical analysis (StatView): Student's t-test onmaximum tumor size. P-values < 0.05 are considered as significant.

Unpaired t-test for Largest Tumor size Grouping Variable: groupHypothesized Difference = 0 Mean Diff. DF t-Value P-Value p17-sp3-4,NS3-100-sp3-4 2.950 5 .739 .4933 p17-sp3-4, NS3/4-10-sp3-4 2.033 6 .628.5532 p17-sp3-4, NS3-10-sp3-4 −1.800 8 −.643 .5383 p17-sp3-4,NS3/4-100-sp3-4 4.950 7 1.742 .1250 p17-sp3-4, PBS-sp3-4 −1.300 8 −.442.6700 p17-sp3-4, rNS3-sp3-4 −3.133 6 −.854 .4259 NS3-100-sp3-4,NS3/4-10-sp3-4 −.917 3 −.542 .6254 NS3-100-sp3-4, NS3-10-sp3-4 −4.750 5−1.811 .1299 NS3-100-sp3-4, NS3/4-100-sp3-4 2.000 4 1.092 .3360NS3-100-sp3-4, PBS-sp3-4 −4.250 5 −1.408 .2183 NS3-100-sp3-4, rNS3-sp3-4−6.083 3 −1.744 .1795 NS3/4-10-sp3-4, NS3-10-sp3-4 −3.833 6 −1.763 .1283NS3/4-10-sp3-4, NS3/4-100-sp3-4 2.917 5 1.824 .1277 NS3/4-10-sp3-4,PBS-sp3-4 −3.333 6 −1.344 .2274 NS3/4-10-sp3-4, rNS3-sp3-4 −5.167 4−1.830 .1412 NS3-10-sp3-4, NS3/4-100-sp3-4 6.750 7 3.416 .0112NS3-10-sp3-4, PBS-sp3-4 .500 8 .215 .8350 NS3-10-sp3-4, rNS3-sp3-4−1.333 6 −.480 .6480 NS3/4-100-sp3-4, PBS-sp3-4 −6.250 7 −2.814 .0260NS3/4-100-sp3-4, rNS3-sp3-4 −8.083 5 −3.179 .0246 PBS-sp3-4, rNS3-sp3-4−1.833 6 −.607 .5662

The example below describes more experiments that were performed todetermine whether the reduction in tumor size can be attributed to thegeneration of NS3-specific T-lymphocytes.

Example 6

In the next set of experiments, the inhibition of SP2/0 or NS3/4A-SP2/0tumor growth was again evaluated in NS3/4A-pVAX immunized Balb/c mice.In mice immunized with the NS3/4A-pVAX plasmid, the growth ofNS3/4A-SP2/0 tumor cells was significantly inhibited as compared togrowth of the non-transfected SP2/0 cells. (See TABLE 7). Thus,NS3/4A-pVAX immunization elicits CTLs that inhibit growth of cellsexpressing NS3/4A in vivo.

TABLE 7 Maximum Mouse Dose Tumor Tumor tumor size ID Immunogen (μg) cellline growth (mm) 11 NS3/4A-pVAX 100 SP2/0 No — 12 NS3/4A-pVAX 100 SP2/0Yes 24 13 NS3/4A-pVAX 100 SP2/0 Yes 9 14 NS3/4A-pVAX 100 SP2/0 Yes 11 15NS3/4A-pVAX 100 SP2/0 Yes 25 4/5 17.25 ± 8.421 16 NS3/4A-pVAX 100NS3/4A-SP2/0 No — 17 NS3/4A-pVAX 100 NS3/4A-SP2/0 Yes 9 18 NS3/4A-pVAX100 NS3/4A-SP2/0 Yes 7 19 NS3/4A-pVAX 100 NS3/4A-SP2/0 Yes 5 20NS3/4A-pVAX 100 NS3/4A-SP2/0 Yes 4 4/5  6.25 ± 2.217 Note: Statisticalanalysis (StatView): Student's t-test on maximum tumor size. P-values <0.05 are considered significant.

Unpaired t-test for Max diam Grouping Variable: Column 1 HypothesizedDifference = 0 Row exclusion: NS3DNA-Tumor-001213 Mean Diff. DF t-ValueP-Value NS3/4-sp2, NS3/4-spNS3 11.000 6 2.526 0.044

Group Info for Max diam Grouping Variable: Column 1 Row exclusion:NS3DNA-Tumor-001213 Count Mean Variance Std. Dev. Std. Err NS3/4-sp2 417.250 70.917 8.421 4.211 NS3/4-spNS3 4 6.250 4.917 2.217 1.109

In another set of experiments, the inhibition of NS3/4A-expressing SP2/0tumor growth was evaluated in MSLF1-pVAX immunized Balb/c mice. Inbrief, groups of mice were immunized with different immunogens (4 μg ofplasmid) using a gene gun at weeks zero, four, eight, twelve, andsixteen. Two weeks after the last immunization approximately 2×10⁶NS3/4A-expressing SP2/0 cells were injected s.c into the right flank ofthe mouse. The kinetics of the tumor growth was then monitored bymeasuring the tumor size through the skin at days seven, 11, and 13. Themean tumor sizes were calculated and groups were compared using theMann-Whitney non-parametric test. At day 14 all mice were sacrificed.

After only a single immunization, tumor inhibiting responses wereobserved. (See FIG. 2 and TABLE 8). After two immunizations, both theNS3/4A-pVAX and MSLF1-pVAX plasmids primed tumor-inhibiting responses.(See FIG. 3 and TABLE 9). The tumors were significantly smaller in miceimmunized with the MSLF1 gene, however, as compared to the native NS3/4Agene. After three injections, both plasmids effectively primedcomparable tumor inhibiting responses. (See FIG. 4 and TABLE 10). Theseexperiments provided evidence that the MSLF-1 gene was more efficient inactivating tumor inhibiting immune responses in vivo than NS3/4A-pVAX.

TABLE 8 Non- Group MSLF1-pVAX1 NS3/4A-pVAX1 immunized MSLF1-pVAX1 — N.S.p < 0.05 NS3/4A-pVAX1 N.S. — p < 0.05 Non- p < 0.05 p < 0.05 — immunized

TABLE 9 Non- Group MSLF1-pVAX1 NS3/4A-pVAX1 immunized MSLF1-pVAX1 — p <0.05 p < 0.01 NS3/4A-pVAX1 p < 0.05 — p < 0.01 Non- p < 0.01 p < 0.01 —immunized

TABLE 10 Non- Group MSLF1-pVAX1 NS3/4A-pVAX1 immunized MSLF1-pVAX1 —N.S. p < 0.01 NS3/4A-pVAX1 N.S. — p < 0.01 Non- p < 0.01 p < 0.01 —immunized

The example below describes experiments that were performed to analyzethe efficiency of various NS3 containing compositions in eliciting acell-mediated response to NS3.

Example 7

To determine whether NS3-specific T-cells were elicited by the NS3/4Aimmunizations, an in vitro T-cell mediated tumor cell lysis assay wasemployed. The assay has been described in detail previously (Sallberg etal., J. Virol. 71:5295 (1997), herein expressly incorporated byreference in its entirety). In a first set of experiments, groups offive Balb/c mice were immunized three times with 100 μg NS3/4A-pVAX i.m.Two weeks after the last injection the mice were sacrificed andsplenocytes were harvested. Re-stimulation cultures with 3×10⁶splenocytes and 3×10⁶ NS3/4A-SP2/0 cells were set. After five days, astandard Cr⁵¹-release assay was performed using NS3/4A-SP2/0 or SP2/0cells as targets. Percent specific lysis was calculated as the ratiobetween lysis of NS3/4A-SP2/0 cells and lysis of SP2/0 cells. Miceimmunized with NS3/4A-pVAX displayed specific lysis over 10% in four outof five tested mice, using an effector to target ratio of 20:1 (SeeFIGS. 5A and 5B).

In a next set of experiments, the T cell responses to MSLF1-pVAX andNS3/4A-pVAX were compared. The ability of the two plasmids to prime invitro detectable CTLs were evaluated in C57/BL6 mice since anH-2b-restricted NS3 epitope had been previously mapped. Groups of micewere immunized with the two plasmids and CTLs were detected in vitrousing either peptide coated H-2b expressing RMA-S cells orNS3/4A-expressing EL-4 cells. Briefly, in vitro stimulation was carriedout for five days in 25-ml flasks at a final volume of 12 ml, containing5 U/ml recombinant murine IL-2 (mIL-2; R&D Systems, Minneapolis, Minn.).The restimulation culture contained a total of 40×10⁶ immune spleencells and 2×10⁶ irradiated (10,000 rad) syngenic SP2/0 cells expressingthe NS3/4A protein. After five days in vitro stimulation a standard⁵¹Cr-release assay was performed. Effector cells were harvested and afour-hour ⁵¹Cr assay was performed in 96-well U-bottom plates in a totalvolume of 200 μl. A total of 1×10⁶ target cells was labeled for one hourwith 20 μl of ⁵¹Cr (5 mCi/ml) and then washed three times in PBS.Cytotoxic activity was determined at effector:target (E:T) ratios of40:1, 20:1, and 10:1, using 5×10³ ⁵¹Cr-labeled target cells/well.

Alternatively, spleenocytes were harvested from C57BL/6 mice 12 daysafter peptide immunization and were resuspended in RPMI 1640 mediumsupplemented with 10% FCS, 2 mM L-Glutamine, 10 mM HEPES, 100 U/mlPenicillin and 100 μg/ml Streptomycin, 1 mM non-essential amino acids,50 μM β-mercaptoethanol, 1 mM sodium pyruvate. In vitro stimulation wascarried out for five days in 25 ml flasks in a total volume of 12 ml,containing 25×10⁶ spleen cells and 25×10⁶ irradiated (2,000 rad)syngeneic splenocytes. The restimulation was performed in the presenceof 0.05 μM NS3/4A H-2D^(b) binding peptide (sequence GAVQNEVTL SEQ. ID.NO.: 37) or a control peptide H-2D^(b) peptide (sequence KAVYNFATM SEQ.ID. NO.: 38). After five days a ⁵¹Cr-release assay was performed. RMA-Starget cells were pulsed with 50 μM peptide for 1.5 hrs at +37° C. priorto ⁵¹Cr-labelling, and then washed three times in PBS. Effector cellswere harvested and the four hour ⁵¹Cr assay was performed as described.Cytotoxic activity was determined at the E:T ratios 60:1, 20:1, and 7:1with 5×10³ ⁵¹Cr-labeled target cells/well. By these assays, it wasdetermined that the MSLF 1 gene primed higher levels of in vitro lyticactivity compared to the NS3/4A-pVAX vector. (See FIG. 6A-6L). Similarresults were obtained with both the peptide coated H-2b expressing RMA-Scells and NS3/4A-expressing EL-4 cells.

Additional evidence that the codon-optimized MSLF1 gene primedNS3-specific CTLs more effectively than the native NS3/4A gene wasobtained using flow cytometry. The frequency of NS3/4A-peptide specificCD8+ T cells were analyzed by ex-vivo staining of spleen cells fromNS3/4A DNA immunized mice with recombinant soluble dimeric mouseH-2D^(b):Ig fusion protein. Many of the monoclonal antibodies and MHC:Igfusion proteins described herein were purchased from BDB Pharmingen (SanDiego, Calif.); Anti-CD16/CD32 (Fc-block™, clone 2.4G2), FITC conjugatedanti-CD8 (clone 53-6.7), FITC conjugated anti-H-2K^(b) (clone AF6-88.5),FITC conjugated anti-H-2D^(b) (clone KH95), recombinant soluble dimericmouse H-2D^(b):Ig, PE conjugated Rat-α Mouse IgG1 (clone X56).

Approximately, 2×10⁶ spleen cells resuspended in 100 μl PBS/1% FCS (FACSbuffer) were incubated with 1 μg/10⁶ cells of Fc-blocking antibodies onice for 15 minutes. The cells were then incubated on ice for 1.5 hrswith either 2 μg/10⁶ cells of H-2D^(b):Ig preloaded for 48 hours at +4°C. with 640 nM excess of NS3/4A derived peptide (sequence GAVQNEVTL SEQ.ID. NO.: 37) or 2 μg/10⁶ cells of unloaded H-2D^(b):Ig fusion protein.The cells were then washed twice in FACS buffer and resuspended in 100μl FACS buffer containing 10 μl/100 μl PE conjugated Rat-α Mouse IgG1secondary antibody and incubated on ice for 30 minutes. The cells werethen washed twice in FACS buffer and incubated with 1 μg/10⁶ cells ofFITC conjugated α-mouse CD8 antibody for 30 minutes. The cells were thenwashed twice in FACS buffer and resuspended in 0.5 ml FACS buffercontaining 0.5 μg/ml of PI. Approximately 200,000 events from eachsample were acquired on a FACS Calibur (BDB) and dead cells (PI positivecells) were excluded from the analysis.

The advantage of quantifying specific CTLs by FACS analysis is that itbypasses the possible disadvantages of in vitro expansion of CTLs invitro prior to analysis. Direct ex-vivo quantification of NS3-specificCTLs using NS3-peptide loaded divalent H-2D^(b):Ig fusion proteinmolecules revealed that the codon optimized MSLF-1 gene primed aeffectively primed NS3-specific CTLs already after two immunizations,whereas the original NS3/4A gene did not (Table). Thus, the optimizedMSLF-1 gene effectively primes NS3-specific CTLs that are of higherfrequency and of better functionality by all parameters tested, ascompared to the original NS3/4A gene.

The next section describes some of the peptide embodiments of theinvention.

HCV Peptides

The embodied HCV peptides or derivatives thereof, include but are notlimited to, those containing as a primary amino acid sequence all of theamino acid sequence substantially as depicted in the Sequence Listing(SEQ. ID. NOs.: 2-11 and SEQ. ID. NO.: 36) and fragments of SEQ. ID.NOs.: 2-11 and SEQ. ID. NO.: 36 that are at least four amino acids inlength (e.g., SEQ. ID. NOs.: 14-16) including altered sequences in whichfunctionally equivalent amino acid residues are substituted for residueswithin the sequence resulting in a silent change. Preferred fragments ofa sequence of SEQ. ID. NOs.: 2-11 and SEQ. ID. NO.: 36 are at least fouramino acids and comprise amino acid sequence unique to the discoveredNS3/4A peptide or mutants thereof including altered sequences in whichfunctionally equivalent amino acid residues are substituted for residueswithin the sequence resulting in a silent change. The HCV peptides canbe, for example, at least 12-704 amino acids in length (e.g., any numberbetween 12-15, 15-20, 20-25, 25-50, 50-100, 100-150, 150-250, 250-500 or500-704 amino acids in length).

Embodiments also include HCV peptides that are substantially identicalto those described above. That is, HCV peptides that have one or moreamino acid residues within SEQ. ID. NOs.: 2-11 and SEQ. ID. NO.: 36 andfragments thereof that are substituted by another amino acid of asimilar polarity that acts as a functional equivalent, resulting in asilent alteration. Further, the HCV peptides can have one or more aminoacid residues fused to SEQ. ID. NOs.: 2-11 and SEQ. ID. NO.: 36 or afragment thereof so long as the fusion does not significantly alter thestructure or function (e.g., immunogenic properties) of the HCV peptide.Substitutes for an amino acid within the sequence can be selected fromother members of the class to which the amino acid belongs. For example,the non-polar (hydrophobic) amino acids include alanine, leucine,isoleucine, valine, proline, phenylalanine, tryptophan, and methionine.The polar neutral amino acids include glycine, serine, threonine,cysteine, tyrosine, asparagine and glutamine. The positively charged(basic) amino acids include arginine, lysine, and histidine. Thenegatively charged (acidic) amino acids include aspartic acid andglutamic acid. The aromatic amino acids include phenylalanine,tryptophan, and tyrosine. Accordingly, the peptide embodiments of theinvention are said to be consisting essentially of SEQ. ID. NOs.: 2-27and SEQ. ID. NO.: 36 in light of the modifications described above.

The HCV peptides described herein can be prepared by chemical synthesismethods (such as solid phase peptide synthesis) using techniques knownin the art such as those set forth by Merrifield et al., J. Am. Chem.Soc. 85:2149 (1964), Houghten et al., Proc. Natl. Acad. Sci. USA,82:51:32 (1985), Stewart and Young (Solid phase peptide synthesis,Pierce Chem. Co., Rockford, Ill. (1984), and Creighton, 1983, Proteins:Structures and Molecular Principles, W. H. Freeman & Co., N.Y. Suchpolypeptides can be synthesized with or without a methionine on theamino terminus. Chemically synthesized HCV peptides can be oxidizedusing methods set forth in these references to form disulfide bridges.

While the HCV peptides described herein can be chemically synthesized,it can be more effective to produce these polypeptides by recombinantDNA technology. Such methods can be used to construct expression vectorscontaining the HCV nucleotide sequences described above, for example,and appropriate transcriptional and translational control signals. Thesemethods include, for example, in vitro recombinant DNA techniques,synthetic techniques, and in vivo genetic recombination. Alternatively,RNA capable of encoding an HCV nucleotide sequence can be chemicallysynthesized using, for example, synthesizers. See, for example, thetechniques described in Oligonucleotide Synthesis, 1984, Gait, M. J.ed., IRL Press, Oxford. Accordingly, several embodiments concern celllines that have been engineered to express the embodied HCV peptides.For example, some cells are made to express the HCV peptides of SEQ. ID.NOs.: 2-11 and SEQ. ID. NO.: 36 or fragments of these molecules (e.g.,SEQ. ID. NOs.: 14-26).

A variety of host-expression vector systems can be utilized to expressthe embodied HCV peptides. Suitable expression systems include, but arenot limited to, microorganisms such as bacteria (e.g., E. coli or B.subtilis) transformed with recombinant bacteriophage DNA, plasmid DNA orcosmid DNA expression vectors containing HCV nucleotide sequences; yeast(e.g., Saccharomyces, Pichia) transformed with recombinant yeastexpression vectors containing the HCV nucleotide sequences; insect cellsystems infected with recombinant virus expression vectors (e.g.,baculovirus) containing the HCV sequences; plant cell systems infectedwith recombinant virus expression vectors (e.g., cauliflower mosaicvirus, CaMV; tobacco mosaic virus, TMV) or transformed with recombinantplasmid expression vectors (e.g., Ti plasmid) containing HCV sequences;or mammalian cell systems (e.g., COS, CHO, BHK, 293, 3T3) harboringrecombinant expression constructs containing promoters derived from thegenome of mammalian cells (e.g., metallothionein promoter) or frommammalian viruses (e.g., the adenovirus late promoter; the vacciniavirus 7.5K promoter).

In bacterial systems, a number of expression vectors can beadvantageously selected depending upon the use intended for the HCV geneproduct being expressed. For example, when a large quantity of such aprotein is to be produced, for the generation of pharmaceuticalcompositions of HCV peptide or for raising antibodies to the HCVpeptide, for example, vectors which direct the expression of high levelsof fusion protein products that are readily purified can be desirable.Such vectors include, but are not limited, to the E. coli expressionvector pUR278 (Ruther et al., EMBO J., 2:1791 (1983), in which the HCVcoding sequence can be ligated individually into the vector in framewith the lacZ coding region so that a fusion protein is produced; pINvectors (Inouye & Inouye, Nucleic Acids Res., 13:3101-3109 (1985); VanHeeke & Schuster, J. Biol. Chem., 264:5503-5509 (1989)); and the like.The pGEX vectors can also be used to express foreign polypeptides asfusion proteins with glutathione S-transferase (GST). In general, suchfusion proteins are soluble and can be purified from lysed cells byadsorption to glutathione-agarose beads followed by elution in thepresence of free glutathione. The PGEX vectors are designed to includethrombin or factor Xa protease cleavage sites so that the cloned targetgene product can be released from the GST moiety.

In an insect system, Autographa californica nuclear polyhedrosis virus(AcNPV) is used as a vector to express foreign genes. The virus grows inSpodoptera frugiperda cells. The HCV coding sequence can be clonedindividually into non-essential regions (for example the polyhedringene) of the virus and placed under control of an AcNPV promoter (forexample the polyhedrin promoter). Successful insertion of an HCV genecoding sequence will result in inactivation of the polyhedrin gene andproduction of non-occluded recombinant virus, (i.e., virus lacking theproteinaceous coat coded for by the polyhedrin gene). These recombinantviruses are then used to infect Spodoptera frupperda cells in which theinserted gene is expressed. (See e.g., Smith et al., J. Virol. 46: 584(1983); and Smith, U.S. Pat. No. 4,215,051, herein expresslyincorporated by reference in its entirety).

In mammalian host cells, a number of viral-based expression systems canbe utilized. In cases where an adenovirus is used as an expressionvector, the HCV nucleotide sequence of interest can be ligated to anadenovirus transcription/translation control complex, e.g., the latepromoter and tripartite leader sequence. This chimeric gene can then beinserted in the adenovirus genome by in vitro or in vivo recombination.Insertion in a non-essential region of the viral genome (e.g., region E1or E3) will result in a recombinant virus that is viable and capable ofexpressing the HCV gene product in infected hosts. (See e.g., Logan &Shenk, Proc. Natl. Acad. Sci. USA 81:3655-3659 (1984)). Specificinitiation signals can also be required for efficient translation ofinserted HCV nucleotide sequences. These signals include the ATGinitiation codon and adjacent sequences.

However, in cases where only a portion of the HCV coding sequence isinserted, exogenous translational control signals, including, perhaps,the ATG initiation codon, can be provided. Furthermore, the initiationcodon can be in phase with the reading frame of the desired codingsequence to ensure translation of the entire insert. These exogenoustranslational control signals and initiation codons can be of a varietyof origins, both natural and synthetic. The efficiency of expression canbe enhanced by the inclusion of appropriate transcription enhancerelements, transcription terminators, etc. (See Bittner et al., Methodsin Enzymol., 153:516-544 (1987)).

In addition, a host cell strain can be chosen, which modulates theexpression of the inserted sequences, or modifies and processes the geneproduct in the specific fashion desired. Such modifications (e.g.,glycosylation) and processing (e.g., cleavage) of protein products areimportant for the function of the protein. Different host cells havecharacteristic and specific mechanisms for the post-translationalprocessing and modification of proteins and gene products. Appropriatecell lines or host systems can be chosen to ensure the correctmodification and processing of the foreign protein expressed. To thisend, eukaryotic host cells that possess the cellular machinery forproper processing of the primary transcript, glycosylation, andphosphorylation of the gene product can be used. Such mammalian hostcells include, but are not limited to, CHO, VERO, BHK, HeLa, COS, MDCK,293, 3T3, and WI38.

For long-term, high-yield production of recombinant proteins, stableexpression is preferred. For example, cell lines that stably express theHCV peptides described above can be engineered. Rather than usingexpression vectors that contain viral origins of replication, host cellscan be transformed with DNA controlled by appropriate expression controlelements (e.g., promoter, enhancer sequences, transcription terminators,polyadenylation sites, etc.), and a selectable marker. Following theintroduction of the foreign DNA, engineered cells are allowed to growfor 1-2 days in an enriched media, and then are switched to a selectivemedia. The selectable marker in the recombinant plasmid confersresistance to the selection and allows cells to stably integrate theplasmid into their chromosomes and grow to form foci which in turn arecloned and expanded into cell lines. This method is advantageously usedto engineer cell lines which express the HCV gene product.

A number of selection systems can be used, including but not limited tothe herpes simplex virus thymidine kinase (Wigler, et al., Cell 11:223(1977), hypoxanthine-guanine phosphoribosyltransferase (Szybalska &Szybalski, Proc. Natl. Acad. Sci. USA 48:2026 (1962)), and adeninephosphoribosyltransferase (Lowy, et al., Cell 22:817 (1980)) genes canbe employed in tk⁻, hgprt⁻ or aprt⁻ cells, respectively. Also,antimetabolite resistance can be used as the basis of selection for thefollowing genes: dhfr, which confers resistance to methotrexate (Wigler,et al., Proc. Natl. Acad. Sci. USA 77:3567 (1980); O'Hare, et al., Proc.Natl. Acad. Sci. USA 78:1527 (1981)); gpt, which confers resistance tomycophenolic acid (Mulligan & Berg, Proc. Natl. Acad. Sci. USA 78:2072(1981)); neo, which confers resistance to the aminoglycoside G-418(Colberre-Garapin, et al., J. Mol. Biol. 150:1 (1981)); and hygro, whichconfers resistance to hygromycin (Santerre, et al., Gene 30:147 (1984)).

Alternatively, any fusion protein can be readily purified by utilizingan antibody specific for the fusion protein being expressed. Forexample, a system described by Janknecht et al. allows for the readypurification of non-denatured fusion proteins expressed in human celllines. (Janknecht, et al., Proc. Natl. Acad. Sci. USA 88: 8972-8976(1991)). In this system, the gene of interest is subcloned into avaccinia recombination plasmid such that the gene's open reading frameis translationally fused to an amino-terminal tag consisting of sixhistidine residues. Extracts from cells infected with recombinantvaccinia virus are loaded onto Ni²⁺ nitriloacetic acid-agarose columnsand histidine-tagged proteins are selectively eluted withimidazole-containing buffers. The example below describes a method thatwas used to express the HCV peptides encoded by the embodied nucleicacids.

Example 8

To characterize NS3/4A-pVAX, MSLF1-pVAX, and the NS3/4A mutantconstructs, described in Example 1, the plasmids were transcribed andtranslated in vitro, and the resulting polypeptides were visualized bysodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Invitro transcription and translation were performed using the T7 coupledreticulocyte lysate system (Promega, Madison, Wis.) according to themanufacturer's instructions. All in vitro translation reactions of theexpression constructs were carried out at 30° C. with ³⁵S-labeledmethionine (Amersham International, Plc, Buckinghamshire, UK). Thelabeled proteins were separated by 12% SDS-PAGE and visualized byexposure to X-ray film (Hyper Film-MP, Amersham) for 6-18 hours.

The in vitro analysis revealed that all proteins were expressed to highamounts from their respective expression constructs. The rNS3 construct(NS3-pVAX vector) produced a single peptide of approximately 61 kDa,whereas, the mutant constructs (e.g., the TGT construct(NS3/4A-TGT-pVAX) and the RGT construct (NS3/4A-RGT-pVAX)) produced asingle polypeptide of approximately 67 kDa, which is identical to themolecular weight of the uncleaved NS3/4A peptide produced from theNS3/4A-pVAX construct. The cleaved product produced from the expressedNS3/4A peptide was approximately 61 kDa, which was identical in size tothe rNS3 produced from the NS3-pVAX vector. These results demonstratedthat the expression constructs were functional, the NS3/4A construct wasenzymatically active, the rNS3 produced a peptide of the predicted size,and the breakpoint mutations completely abolished cleavage at theNS3-NS4A junction.

To compare the translation efficiency from the NS3/4A-pVAX andMSLF1-pVAX plasmids, the amount of input DNA was serially diluted priorto addition to the assay. Serial dilutions of the plasmids revealed thatthe MSLF1 plasmid gave stronger bands at higher dilutions of the plasmidthan the wild-type NS3/4A plasmid, providing evidence that in vitrotranscription and translation was more efficient from the MSLF 1plasmid. The NS3/4A-pVAX and MSLF1 plasmids were then analyzed forprotein expression using transiently transfected Hep-G2 cells. Similarresults were obtained in that the MSLF-1 gene provided more efficientexpression of NS3 than the native NS3/4A gene.

The sequences, constructs, vectors, clones, and other materialscomprising the embodied HCV nucleic acids and peptides can be inenriched or isolated form. As used herein, “enriched” means that theconcentration of the material is many times its natural concentration,for example, at least about 2, 5, 10, 100, or 1000 times its naturalconcentration, advantageously 0.01%, by weight, preferably at leastabout 0.1% by weight. Enriched preparations from about 0.5% or more, forexample, 1%, 5%, 10%, and 20% by weight are also contemplated. The term“isolated” requires that the material be removed from its originalenvironment (e.g., the natural environment if it is naturallyoccurring). For example, a naturally-occurring polynucleotide present ina living animal is not isolated, but the same polynucleotide, separatedfrom some or all of the coexisting materials in the natural system, isisolated. It is also advantageous that the sequences be in purifiedform. The term “purified” does not require absolute purity; rather, itis intended as a relative definition. Isolated proteins have beenconventionally purified to electrophoretic homogeneity by Coomassiestaining, for example. Purification of starting material or naturalmaterial to at least one order of magnitude, preferably two or threeorders, and more preferably four or five orders of magnitude isexpressly contemplated.

The HCV gene products described herein can also be expressed in plants,insects, and animals so as to create a transgenic organism. Desirabletransgenic plant systems having an HCV peptide include Arabadopsis,maize, and Chlamydomonas. Desirable insect systems having an HCV peptideinclude, but are not limited to, D. melanogaster and C. elegans. Animalsof any species, including, but not limited to, amphibians, reptiles,birds, mice, hamsters, rats, rabbits, guinea pigs, pigs, micro-pigs,goats, dogs, cats, and non-human primates, e.g., baboons, monkeys, andchimpanzees can be used to generate transgenic animals having anembodied HCV molecule. These transgenic organisms desirably exhibitgermline transfer of HCV peptides described herein.

Any technique known in the art is preferably used to introduce the HCVtransgene into animals to produce the founder lines of transgenicanimals or to knock out or replace existing HCV genes. Such techniquesinclude, but are not limited to pronuclear microinjection (Hoppe, P. C.and Wagner, T. E., 1989, U.S. Pat. No. 4,873,191); retrovirus mediatedgene transfer into germ lines (Van der Putten et al., Proc. Natl. Acad.Sci., USA 82:6148-6152 (1985)); gene targeting in embryonic stem cells(Thompson et al., Cell 56:313-321 (1989)); electroporation of embryos(Lo, Mol. Cell. Biol. 3:1803-1814 (1983); and sperm-mediated genetransfer (Lavitrano et al., Cell 57:717-723 (1989)); see also Gordon,Transgenic Animals, Intl. Rev. Cytol. 115:171-229 (1989).

Following synthesis or expression and isolation or purification of theHCV peptides, the isolated or purified peptide can be used to generateantibodies. Depending on the context, the term “antibodies” canencompass polyclonal, monoclonal, chimeric, single chain, Fab fragmentsand fragments produced by a Fab expression library. Antibodies thatrecognize the HCV peptides have many uses including, but not limited to,biotechnological applications, therapeutic/prophylactic applications,and diagnostic applications.

For the production of antibodies, various hosts including goats,rabbits, rats, mice, and humans etc. can be immunized by injection withan HCV peptide. Depending on the host species, various adjuvants can beused to increase immunological response. Such adjuvants include, but arenot limited to, ribavirin, Freund's, mineral gels such as aluminumhydroxide, and surface active substances such as lysolecithin, pluronicpolyols, polyanions, peptides, oil emulsions, keyhole limpet hemocyanin,and dinitrophenol. BCG (Bacillus Calmette-Guerin) and Corynebacteriumparvum are also potentially useful adjuvants.

Peptides used to induce specific antibodies can have an amino acidsequence consisting of at least four amino acids, and preferably atleast 10 to 15 amino acids. By one approach, short stretches of aminoacids encoding fragments of NS3/4A are fused with those of anotherprotein such as keyhole limpet hemocyanin such that an antibody isproduced against the chimeric molecule. Additionally, a compositioncomprising ribavirin and an HCV peptide (SEQ. ID. NOs.: 2-11 and SEQ.ID. NO.: 36), a fragment thereof containing any number of consecutiveamino acids between at least 3-50 (e.g., 3, 4, 6, 8, 10, 12, 15, 20, 25,30, 35, 40, 45, or 50 amino acids) (e.g., SEQ. ID. NOs.: 4-26), or anucleic acid encoding one or more of these molecules is administered toan animal, preferably a mammal including a human. While antibodiescapable of specifically recognizing HCV can be generated by injectingsynthetic 3-mer, 10-mer, and 15-mer peptides that correspond to an HCVpeptide into mice, a more diverse set of antibodies can be generated byusing recombinant HCV peptides, prepared as described above.

To generate antibodies to an HCV peptide, substantially pure peptide isisolated from a transfected or transformed cell. The concentration ofthe peptide in the final preparation is adjusted, for example, byconcentration on an Amicon filter device, to the level of a fewmicrograms/ml. Monoclonal or polyclonal antibody to the peptide ofinterest can then be prepared as follows:

Monoclonal antibodies to an HCV peptide can be prepared using anytechnique that provides for the production of antibody molecules bycontinuous cell lines in culture. These include, but are not limited to,the hybridoma technique originally described by Koehler and Milstein(Nature 256:495-497 (1975)), the human B-cell hybridoma technique(Kosbor et al. Immunol Today 4:72 (1983)); Cote et al Proc Natl Acad Sci80:2026-2030 (1983), and the EBV-hybridoma technique Cole et al.Monoclonal Antibodies and Cancer Therapy, Alan R. Liss Inc, New YorkN.Y., pp 77-96 (1985). In addition, techniques developed for theproduction of “chimeric antibodies”, the splicing of mouse antibodygenes to human antibody genes to obtain a molecule with appropriateantigen specificity and biological activity can be used. (Morrison etal. Proc Natl Acad Sci 81:6851-6855 (1984); Neuberger et al. Nature312:604-608 (1984); Takeda et al. Nature 314:452-454 (1985)).Alternatively, techniques described for the production of single chainantibodies (U.S. Pat. No. 4,946,778) can be adapted to produceHCV-specific single chain antibodies. Antibodies can also be produced byinducing in vivo production in the lymphocyte population or by screeningrecombinant immunoglobulin libraries or panels of highly specificbinding reagents as disclosed in Orlandi et al., Proc Natl Acad Sci 86:3833-3837 (1989), and Winter G. and Milstein C; Nature 349:293-299(1991).

Antibody fragments that contain specific binding sites for an HCVpeptide can also be generated. For example, such fragments include, butare not limited to, the F(ab′)₂ fragments that can be produced by pepsindigestion of the antibody molecule and the Fab fragments that can begenerated by reducing the disulfide bridges of the F(ab′)₂ fragments.Alternatively, Fab expression libraries can be constructed to allowrapid and easy identification of monoclonal Fab fragments with thedesired specificity. (Huse W. D. et al. Science 256:1275-1281 (1989)).

By one approach, monoclonal antibodies to an HCV peptide are made asfollows. Briefly, a mouse is repetitively inoculated with a fewmicrograms of the selected protein or peptides derived therefrom over aperiod of a few weeks. The mouse is then sacrificed, and the antibodyproducing cells of the spleen isolated. The spleen cells are fused inthe presence of polyethylene glycol with mouse myeloma cells, and theexcess unfused cells destroyed by growth of the system on selectivemedia comprising aminopterin (HAT media). The successfully fused cellsare diluted and aliquots of the dilution placed in wells of a microtiterplate where growth of the culture is continued. Antibody-producingclones are identified by detection of antibody in the supernatant fluidof the wells by immunoassay procedures, such as ELISA, as originallydescribed by Engvall, E., Meth. Enzymol. 70:419 (1980), and derivativemethods thereof. Selected positive clones can be expanded and theirmonoclonal antibody product harvested for use. Detailed procedures formonoclonal antibody production are described in Davis, L. et al. BasicMethods in Molecular Biology Elsevier, New York. Section 21-2.

Polyclonal antiserum containing antibodies to heterogeneous epitopes ofa single protein can be prepared by immunizing suitable animals with theexpressed protein or peptides derived therefrom described above, whichcan be unmodified or modified to enhance immunogenicity. Effectivepolyclonal antibody production is affected by many factors related bothto the antigen and the host species. For example, small molecules tendto be less immunogenic than others and can require the use of carriersand adjuvant. Also, host animals vary in response to site ofinoculations and dose, with both inadequate or excessive doses ofantigen resulting in low titer antisera. Small doses (ng level) ofantigen administered at multiple intradermal sites appears to be mostreliable. An effective immunization protocol for rabbits can be found inVaitukaitis, J. et al. J. Clin. Endocrinol. Metab. 33:988-991 (1971).

Booster injections are given at regular intervals, and antiserumharvested when antibody titer thereof, as determinedsemi-quantitatively, for example, by double immunodiffusion in agaragainst known concentrations of the antigen, begins to fall See, forexample, Ouchterlony, O. et al., Chap. 19 in: Handbook of ExperimentalImmunology D. Wier (ed) Blackwell (1973). Plateau concentration ofantibody is usually in the range of 0.1 to 0.2 mg/ml of serum (about 12μM). Affinity of the antisera for the antigen is determined by preparingcompetitive binding curves, as described, for example, by Fisher, D.,Chap. 42 in: Manual of Clinical Immunology, 2d Ed. (Rose and Friedman,Eds.) Amer. Soc. For Microbiol., Washington, D.C. (1980). Antibodypreparations prepared according to either protocol are useful inquantitative immunoassays that determine concentrations ofantigen-bearing substances in biological samples; they are also usedsemi-quantitatively or qualitatively (e.g., in diagnostic embodimentsthat identify the presence of HCV in biological samples). The nextsection describes how some of the novel nucleic acids and peptidesdescribed above can be used in diagnostics.

Diagnostic Embodiments

Generally, the embodied diagnostics are classified according to whethera nucleic acid or protein-based assay is used. Some diagnostic assaysdetect the presence or absence of an embodied HCV nucleic acid sequencein a sample obtained from a patient, whereas, other assays seek toidentify whether an embodied HCV peptide is present in a biologicalsample obtained from a patient. Additionally, the manufacture of kitsthat incorporate the reagents and methods described herein that allowfor the rapid detection and identification of HCV are also embodied.These diagnostic kits can include, for example, an embodied nucleic acidprobe or antibody, which specifically detects HCV. The detectioncomponent of these kits will typically be supplied in combination withone or more of the following reagents. A support capable of absorbing orotherwise binding DNA, RNA, or protein will often be supplied. Availablesupports include membranes of nitrocellulose, nylon or derivatized nylonthat can be characterized by bearing an array of positively chargedsubstituents. One or more restriction enzymes, control reagents,buffers, amplification enzymes, and non-human polynucleotides likecalf-thymus or salmon-sperm DNA can be supplied in these kits.

Useful nucleic acid-based diagnostics include, but are not limited to,direct DNA sequencing, Southern Blot analysis, dot blot analysis,nucleic acid amplification, and combinations of these approaches. Thestarting point for these analysis is isolated or purified nucleic acidfrom a biological sample obtained from a patient suspected ofcontracting HCV or a patient at risk of contracting HCV. The nucleicacid is extracted from the sample and can be amplified by RT-PCR and/orDNA amplification using primers that correspond to regions flanking theembodied HCV nucleic acid sequences (e.g., NS3/4A (SEQ. ID. NO.: 1)).

In some embodiments, nucleic acid probes that specifically hybridizewith HCV sequences are attached to a support in an ordered array,wherein the nucleic acid probes are attached to distinct regions of thesupport that do not overlap with each other. Preferably, such an orderedarray is designed to be “addressable” where the distinct locations ofthe probe are recorded and can be accessed as part of an assayprocedure. These probes are joined to a support in different knownlocations. The knowledge of the precise location of each nucleic acidprobe makes these “addressable” arrays particularly useful in bindingassays. The nucleic acids from a preparation of several biologicalsamples are then labeled by conventional approaches (e.g., radioactivityor fluorescence) and the labeled samples are applied to the array underconditions that permit hybridization.

If a nucleic acid in the samples hybridizes to a probe on the array,then a signal will be detected at a position on the support thatcorresponds to the location of the hybrid. Since the identity of eachlabeled sample is known and the region of the support on which thelabeled sample was applied is known, an identification of the presenceof the polymorphic variant can be rapidly determined. These approachesare easily automated using technology known to those of skill in the artof high throughput diagnostic or detection analysis.

Additionally, an approach opposite to that presented above can beemployed. Nucleic acids present in biological samples can be disposed ona support so as to create an addressable array. Preferably, the samplesare disposed on the support at known positions that do not overlap. Thepresence of HCV nucleic acids in each sample is determined by applyinglabeled nucleic acid probes that complement nucleic acids, which encodeHCV peptides, at locations on the array that correspond to the positionsat which the biological samples were disposed. Because the identity ofthe biological sample and its position on the array is known, theidentification of a patient that has been infected with HCV can berapidly determined. These approaches are also easily automated usingtechnology known to those of skill in the art of high throughputdiagnostic analysis.

Any addressable array technology known in the art can be employed. Oneparticular embodiment of polynucleotide arrays is known as Genechips™,and has been generally described in U.S. Pat. No. 5,143,854; PCTpublications WO 90/15070 and 92/10092. These arrays are generallyproduced using mechanical synthesis methods or light directed synthesismethods, which incorporate a combination of photolithographic methodsand solid phase oligonucleotide synthesis. (Fodor et al., Science,251:767-777, (1991)). The immobilization of arrays of oligonucleotideson solid supports has been rendered possible by the development of atechnology generally identified as “Very Large Scale Immobilized PolymerSynthesis” (VLSPIS™) in which, typically, probes are immobilized in ahigh density array on a solid surface of a chip. Examples of VLSPIS™technologies are provided in U.S. Pat. Nos. 5,143,854 and 5,412,087 andin PCT Publications WO 90/15070, WO 92/10092 and WO 95/11995, whichdescribe methods for forming oligonucleotide arrays through techniquessuch as light-directed synthesis techniques. In designing strategiesaimed at providing arrays of nucleotides immobilized on solid supports,further presentation strategies were developed to order and display theoligonucleotide arrays on the chips in an attempt to maximizehybridization patterns and diagnostic information. Examples of suchpresentation strategies are disclosed in PCT Publications WO 94/12305,WO 94/11530, WO 97/29212, and WO 97/31256.

A wide variety of labels and conjugation techniques are known by thoseskilled in the art and can be used in various nucleic acid assays. Thereare several ways to produce labeled nucleic acids for hybridization orPCR including, but not limited to, oligolabeling, nick translation,end-labeling, or PCR amplification using a labeled nucleotide.Alternatively, a nucleic acid encoding an HCV peptide can be cloned intoa vector for the production of an mRNA probe. Such vectors are known inthe art, are commercially available, and can be used to synthesize RNAprobes in vitro by addition of an appropriate RNA polymerase such as T7,T3 or SP6 and labeled nucleotides. A number of companies such asPharmacia Biotech (Piscataway N.J.), Promega (Madison Wis.), and U.S.Biochemical Corp (Cleveland Ohio) supply commercial kits and protocolsfor these procedures. Suitable reporter molecules or labels includethose radionuclides, enzymes, fluorescent, chemiluminescent, orchromogenic agents, as well as, substrates, cofactors, inhibitors,magnetic particles and the like.

The presence of an HCV peptide in a protein sample obtained from apatient can also be detected by using conventional assays and theembodiments described herein. For example, antibodies that areimmunoreactive with the disclosed HCV peptides can be used to screenbiological samples for the presence of HCV infection. In preferredembodiments, antibodies that are reactive to the embodied HCV peptidesare used to immunoprecipitate the disclosed HCV peptides from biologicalsamples or are used to react with proteins obtained from a biologicalsample on Western or Immunoblots. Favored diagnostic embodiments alsoinclude enzyme-linked immunosorbant assays (ELISA), radioimmunoassays(MA), immunoradiometric assays (IRMA) and immunoenzymatic assays (IEMA),including sandwich assays using monoclonal and/or polyclonal antibodiesspecific for the disclosed HCV peptides. Exemplary sandwich assays aredescribed by David et al., in U.S. Pat. Nos. 4,376,110 and 4,486,530.Other embodiments employ aspects of the immune-strip technologydisclosed in U.S. Pat. Nos. 5,290,678; 5,604,105; 5,710,008; 5,744,358;and 5,747,274.

In another preferred protein-based diagnostic, the antibodies describedherein are attached to a support in an ordered array, wherein aplurality of antibodies are attached to distinct regions of the supportthat do not overlap with each other. As with the nucleic acid-basedarrays, the protein-based arrays are ordered arrays that are designed tobe “addressable” such that the distinct locations are recorded and canbe accessed as part of an assay procedure. These probes are joined to asupport in different known locations. The knowledge of the preciselocation of each probe makes these “addressable” arrays particularlyuseful in binding assays. For example, an addressable array can comprisea support having several regions to which are joined a plurality ofantibody probes that specifically recognize HCV peptides present in abiological sample and differentiate the isotype of HCV identifiedherein.

By one approach, proteins are obtained from biological samples and arethen labeled by conventional approaches (e.g., radioactivity,colorimetrically, or fluorescently). The labeled samples are thenapplied to the array under conditions that permit binding. If a proteinin the sample binds to an antibody probe on the array, then a signalwill be detected at a position on the support that corresponds to thelocation of the antibody-protein complex. Since the identity of eachlabeled sample is known and the region of the support on which thelabeled sample was applied is known, an identification of the presence,concentration, and/or expression level can be rapidly determined. Thatis, by employing labeled standards of a known concentration of HCVpeptide, an investigator can accurately determine the proteinconcentration of the particular peptide in a tested sample and can alsoassess the expression level of the HCV peptide. Conventional methods indensitometry can also be used to more accurately determine theconcentration or expression level of the HCV peptide. These approachesare easily automated using technology known to those of skill in the artof high throughput diagnostic analysis.

In another embodiment, an approach opposite to that presented above canbe employed. Proteins present in biological samples can be disposed on asupport so as to create an addressable array. Preferably, the proteinsamples are disposed on the support at known positions that do notoverlap. The presence of an HCV peptide in each sample is thendetermined by applying labeled antibody probes that recognize epitopesspecific for the HCV peptide. Because the identity of the biologicalsample and its position on the array is known, an identification of thepresence, concentration, and/or expression level of an HCV peptide canbe rapidly determined.

That is, by employing labeled standards of a known concentration of HCVpeptide, an investigator can accurately determine the concentration ofpeptide in a sample and from this information can assess the expressionlevel of the peptide. Conventional methods in densitometry can also beused to more accurately determine the concentration or expression levelof the HCV peptide. These approaches are also easily automated usingtechnology known to those of skill in the art of high throughputdiagnostic analysis. As detailed above, any addressable array technologyknown in the art can be employed. The next section describes morecompositions that include the HCV nucleic acids and/or HCV peptidesdescribed herein.

Compositions Comprising HCV Nucleic Acids or Peptides

Embodiments of the invention also include NS3/4A fusion proteins ornucleic acids encoding these molecules. For instance, production andpurification of recombinant protein may be facilitated by the additionof auxiliary amino acids to form a “tag”. Such tags include, but are notlimited to, His-6, Flag, Myc and GST. The tags may be added to theC-terminus, N-terminus, or within the NS3/4A amino acid sequence.Further embodiments include NS3/4A fusion proteins with amino or carboxyterminal truncations, or internal deletions, or with additionalpolypeptide sequences added to the amino or carboxy terminal ends, oradded internally. Other embodiments include NS3/4A fusion proteins, ortruncated or mutated versions thereof, where the residues of the NS3/4Aproteolytic cleavage site have been substituted. Such substitutionsinclude, but are not limited to, sequences where the P1′ site is a Ser,Gly, or Pro, or the P1 position is an Arg, or where the P8 to P4′sequence is Ser-Ala-Asp-Leu-Glu-Val-Val-Thr-Ser-Thr-Trp-Val (SEQ. ID.NO.: 15).

More embodiments concern an immunogen comprising the NS3/4A fusionprotein, or a truncated, mutated, or modified version thereof, capableof eliciting an enhanced immune response against NS3. The immunogen canbe provided in a substantially purified form, which means that theimmunogen has been rendered substantially free of other proteins,lipids, carbohydrates or other compounds with which it naturallyassociates.

Some embodiments contain at least one of the HCV nucleic acids or HCVpeptides (e.g., SEQ. ID. NOs.: 1-27, 35, or 36) joined to a support.Preferably, these supports are manufactured so as to create a multimericagent. These multimeric agents provide the HCV peptide or nucleic acidin such a form or in such a way that a sufficient affinity to themolecule is achieved. A multimeric agent having an HCV nucleic acid orpeptide can be obtained by joining the desired molecule to amacromolecular support. A “support” can be a termed a carrier, aprotein, a resin, a cell membrane, a capsid or portion thereof, or anymacromolecular structure used to join or immobilize such molecules.Solid supports include, but are not limited to, the walls of wells of areaction tray, test tubes, polystyrene beads, magnetic beads,nitrocellulose strips, membranes, microparticles such as latexparticles, animal cells, Duracyte®, artificial cells, and others. An HCVnucleic acid or peptide can also be joined to inorganic carriers, suchas silicon oxide material (e.g., silica gel, zeolite, diatomaceous earthor aminated glass) by, for example, a covalent linkage through ahydroxy, carboxy or amino group and a reactive group on the carrier.

In several multimeric agents, the macromolecular support has ahydrophobic surface that interacts with a portion of the HCV nucleicacid or peptide by a hydrophobic non-covalent interaction. In somecases, the hydrophobic surface of the support is a polymer such asplastic or any other polymer in which hydrophobic groups have beenlinked such as polystyrene, polyethylene or polyvinyl. Additionally, HCVnucleic acid or peptide can be covalently bound to carriers includingproteins and oligo/polysaccarides (e.g. cellulose, starch, glycogen,chitosane or aminated sepharose). In these later multimeric agents, areactive group on the molecule, such as a hydroxy or an amino group, isused to join to a reactive group on the carrier so as to create thecovalent bond. Additional multimeric agents comprise a support that hasother reactive groups that are chemically activated so as to attach theHCV nucleic acid or peptide. For example, cyanogen bromide activatedmatrices, epoxy activated matrices, thio and thiopropyl gels,nitrophenyl chloroformate and N-hydroxy succinimide chlorformatelinkages, or oxirane acrylic supports are used. (Sigma).

Carriers for use in the body, (i.e. for prophylactic or therapeuticapplications) are desirably physiological, non-toxic and preferably,non-immunoresponsive. Suitable carriers for use in the body includepoly-L-lysine, poly-D, L-alanine, liposomes, capsids that display thedesired HCV peptide or nucleic acid, and Chromosorb® (Johns-ManvilleProducts, Denver Co.). Ligand conjugated Chromosorb® (Synsorb-Pk) hasbeen tested in humans for the prevention of hemolytic-uremic syndromeand was reported as not presenting adverse reactions. (Armstrong et al.J. Infectious Diseases 171:1042-1045 (1995)). For some embodiments, a“naked” carrier (i.e., lacking an attached HCV nucleic acid or peptide)that has the capacity to attach an HCV nucleic acid or peptide in thebody of a organism is administered. By this approach, a “prodrug-type”therapy is envisioned in which the naked carrier is administeredseparately from the HCV nucleic acid or peptide and, once both are inthe body of the organism, the carrier and the HCV nucleic acid orpeptide are assembled into a multimeric complex.

The insertion of linkers, (e.g., “λ linkers” engineered to resemble theflexible regions of λ phage) of an appropriate length between the HCVnucleic acid or peptide and the support are also contemplated so as toencourage greater flexibility of the HCV peptide, hybrid, or bindingpartner and thereby overcome any steric hindrance that can be presentedby the support. The determination of an appropriate length of linkerthat allows for an optimal cellular response or lack thereof, can bedetermined by screening the HCV nucleic acid or peptide with varyinglinkers in the assays detailed in the present disclosure.

A composite support comprising more than one type of HCV nucleic acid orpeptide is also envisioned. A “composite support” can be a carrier, aresin, or any macromolecular structure used to attach or immobilize twoor more different HCV nucleic acids or peptides. As above, the insertionof linkers, such as λ linkers, of an appropriate length between the HCVnucleic acid or peptide and the support is also contemplated so as toencourage greater flexibility in the molecule and thereby overcome anysteric hindrance that can occur. The determination of an appropriatelength of linker that allows for an optimal cellular response or lackthereof, can be determined by screening the HCV nucleic acid or peptidewith varying linkers in the assays detailed in the present disclosure.

In other embodiments, the multimeric and composite supports discussedabove can have attached multimerized HCV nucleic acids or peptides so asto create a “multimerized-multimeric support” and a“multimerized-composite support”, respectively. A multimerized ligandcan, for example, be obtained by coupling two or more HCV nucleic acidsor peptides in tandem using conventional techniques in molecularbiology. The multimerized form of the HCV nucleic acid or peptide can beadvantageous for many applications because of the ability to obtain anagent with a higher affinity, for example. The incorporation of linkersor spacers, such as flexible λ linkers, between the individual domainsthat make-up the multimerized agent can also be advantageous for someembodiments. The insertion of λ linkers of an appropriate length betweenprotein binding domains, for example, can encourage greater flexibilityin the molecule and can overcome steric hindrance. Similarly, theinsertion of linkers between the multimerized HCV nucleic acid orpeptide and the support can encourage greater flexibility and limitsteric hindrance presented by the support. The determination of anappropriate length of linker can be determined by screening the HCVnucleic acids or peptides in the assays detailed in this disclosure.

Embodiments also include vaccine compositions and immunogen preparationscomprising the NS3/4A fusion protein, or a truncated or mutated versionthereof, and, optionally, an adjuvant. The next section describes someof these compositions in greater detail.

Vaccine Compositions and Immunogen Preparations

Vaccine compositions and immunogen preparations comprising, consistingof, or consisting essentially of either an embodied HCV nucleic acid orHCV peptide or both (e.g., any one or more of SEQ. ID. NOs.: 1-27, 35 or36) are contemplated. These compositions typically contain an adjuvant,but do not necessarily require an adjuvant. That is many of the nucleicacids and peptides described herein function as immunogens whenadministered neat. The compositions described herein (e.g., the HCVimmunogens and vaccine compositions containing an adjuvant, such asribavirin) can be manufactured in accordance with conventional methodsof galenic pharmacy to produce medicinal agents for administration toanimals, e.g., mammals including humans.

Various nucleic acid-based vaccines are known and it is contemplatedthat these compositions and approaches to immunotherapy can be augmentedby reformulation with ribavirin (See e.g., U.S. Pat. Nos. 5,589,466 and6,235,888). By one approach, for example, a gene encoding one of the HCVpeptides described herein (e.g., SEQ. ID. NO.: 1 or SEQ. ID. NO.: 35) iscloned into an expression vector capable of expressing the polypeptidewhen introduced into a subject. The expression construct is introducedinto the subject in a mixture of adjuvant (e.g., ribavirin) or inconjunction with an adjuvant (e.g., ribavirin). For example, theadjuvant (e.g., ribavirin) is administered shortly after the expressionconstruct at the same site. Alternatively, RNA encoding the HCVpolypeptide antigen of interest is provided to the subject in a mixturewith ribavirin or in conjunction with an adjuvant (e.g., ribavirin).

Where the antigen is to be DNA (e.g., preparation of a DNA vaccinecomposition), suitable promoters include Simian Virus 40 (SV40), MouseMammary Tumor Virus (MMTV) promoter, Human Immunodeficiency Virus (HIV)such as the HIV Long Terminal Repeat (LTR) promoter, Moloney virus, ALV,Cytomegalovirus (CMV) such as the CMV immediate early promoter, EpsteinBarr Virus (EBV), Rous Sarcoma Virus (RSV) as well as promoters fromhuman genes such as human actin, human myosin, human hemoglobin, humanmuscle creatine and human metalothionein can be used. Examples ofpolyadenylation signals useful with some embodiments, especially in theproduction of a genetic vaccine for humans, include but are not limitedto, SV40 polyadenylation signals and LTR polyadenylation signals. Inparticular, the SV40 polyadenylation signal, which is in pCEP4 plasmid(Invitrogen, San Diego Calif.), referred to as the SV40 polyadenylationsignal, is used.

In addition to the regulatory elements required for gene expression,other elements may also be included in a gene construct. Such additionalelements include enhancers. The enhancer may be selected from the groupincluding but not limited to: human actin, human myosin, humanhemoglobin, human muscle creatine and viral enhancers such as those fromCMV, RSV and EBV. Gene constructs can be provided with mammalian originof replication in order to maintain the construct extrachromosomally andproduce multiple copies of the construct in the cell. Plasmids pCEP4 andpREP4 from Invitrogen (San Diego, Calif.) contain the Epstein Barr virusorigin of replication and nuclear antigen EBNA-1 coding region, whichproduces high copy episomal replication without integration. All formsof DNA, whether replicating or non-replicating, which do not becomeintegrated into the genome, and which are expressible, can be used.Preferably, the genetic vaccines comprise ribavirin and a nucleic acidencoding NS3/4A, NS3, or a fragment or mutant thereof (SEQ. ID. NOs.:2-26 and 36). The following example describes the preparation of agenetic vaccine suitable for use in humans.

Example 9

An HCV expression plasmid is designed to express the NS3/4A peptide(SEQ. ID. NO.: 2 or SEQ. ID. NO.: 36). The NS3/4A coding sequence ofNS3/4A-pVAX or MSLF1-pVAX is removed enzymatically, and the isolatedfragment is inserted into plasmid A so that it is under thetranscriptional control of the CMV promoter and the RSV enhancerelement. (See U.S. Pat. No. 6,235,888 to Pachuk, et al.). Plasmidbackbone A is 3969 base pairs in length; it contains a PBR origin ofreplication for replicating in E. coli and a kanamycin resistance gene.Inserts such as the NS3/4A or codon-optimized NS3/4A, are cloned into apolylinker region, which places the insert between and operably linkedto the promoter and polyadenylation signal. Transcription of the clonedinserts is under the control of the CMV promoter and the RSV enhancerelements. A polyadenylation signal is provided by the presence of anSV40 poly A signal situated just 3′ of the cloning site. An NS3/4Acontaining vaccine composition or immunogen preparation is then made bymixing any amount of construct between about 0.5-500 mg, for example,between 0.5-1 μg, 2-5 μg, 5-10 μg, 10-20 μg, 20-50 μg, 50-75 μg, 75-100μg, 100-250 μg, 250 μg-500 μg with any amount of ribavirin between about0.1-10 mg, for example, between 0.1 mg-0.5 mg, 0.5 mg-1 mg, 1 mg-2 mg, 2mg-5 mg, or 5 mg-10 mg of ribavirin.

Said vaccine composition can be used to raise antibodies in a mammal(e.g., mice or rabbits) or can be injected intramuscularly into a humanso as to raise antibodies, preferably a human that is chronicallyinfected with the HCV virus. The recipient preferably receives threeimmunization boosts of the mixture at 4-week intervals, as well. By thethird boost, the titer of antibody specific for HCV will besignificantly increased. Additionally, at this time, said subject willexperience an enhanced antibody and T-cell mediated immune responseagainst NS3, as evidenced by an increased fraction of NS3 specificantibodies as detected by EIA, and a reduction in viral load as detectedby RT-PCR.

Also contemplated are vaccine compositions comprising one or more of theHCV peptides described herein. Preferably, the embodied peptide vaccinescomprise ribavirin and NS3/4A, NS3, or a fragment or mutant thereof(e.g., SEQ. ID. NOs.: 2-26 and 36). The following example describes anapproach to prepare a vaccine composition comprising an NS3/4A fusionprotein and an adjuvant.

Example 10

To generate a tagged NS3/4A construct, the NS3/4A coding sequence ofNS3/4A-pVAX or MSLF1-pVAX is removed enzymatically, and the isolatedfragment is inserted into an Xpress vector (Invitrogen). The Xpressvector allows for the production of a recombinant fusion protein havinga short N-terminal leader peptide that has a high affinity for divalentcations. Using a nickel-chelating resin (Invitrogen), the recombinantprotein can be purified in one step and the leader can be subsequentlyremoved by cleavage with enterokinase. A preferred vector is thepBlueBacHis2 Xpress. The pBlueBacHis2 Xpress vector is a Baculovirusexpression vector containing a multiple cloning site, an ampicillinresistance gene, and a lac z gene. Accordingly, the digestedamplification fragment is cloned into the pBlueBacHis2 Xpress vector andSF9 cells are infected. The expression protein is then isolated orpurified according to the manufacturer's instructions. An NS3/4Acontaining vaccine composition is then made by mixing any amount of therNS3/4A between about 0.1-500 mg, for example, 1-5 μg, 5-10 μg, 10-20μg, 20-30 μg, 30-50 μg, 50-100 μg, 100-250 μg, or 250-500 μg with anyamount of ribavirin between about 0.1-10 mg, for example, between 0.1mg-0.5 mg, 0.5 mg-1 mg, 1 mg-2 mg, 2 mg-5 mg, or 5 mg-10 mg ofribavirin.

Said vaccine composition can be used to raise antibodies in a mammal(e.g., mice or rabbits) or can be injected intramuscularly into a humanso as to raise antibodies, preferably a human that is chronicallyinfected with the HCV virus. The recipient preferably receives threeimmunization boosts of the mixture at 4-week intervals. By the thirdboost, the titer of antibody specific for HCV will be significantlyincreased. Additionally, at this time, said subject will experience anenhanced antibody and T-cell mediated immune response against NS3, asevidenced by an increased fraction of NS3 specific antibodies asdetected by EIA, and a reduction in viral load as detected by RT-PCR.

The compositions that comprise one or more of the embodied HCV nucleicacids or peptides may contain other ingredients including, but notlimited to, adjuvants, binding agents, excipients such as stabilizers(to promote long term storage), emulsifiers, thickening agents, salts,preservatives, solvents, dispersion media, coatings, antibacterial andantifungal agents, isotonic and absorption delaying agents and the like.These compositions are suitable for treatment of animals either as apreventive measure to avoid a disease or condition or as a therapeuticto treat animals already afflicted with a disease or condition.

Many other ingredients can be also be present. For example, the adjuvantand antigen can be employed in admixture with conventional excipients(e.g., pharmaceutically acceptable organic or inorganic carriersubstances suitable for parenteral, enteral (e.g., oral) or topicalapplication that do not deleteriously react with the adjuvent and/orantigen). Suitable pharmaceutically acceptable carriers include, but arenot limited to, water, salt solutions, alcohols, gum arabic, vegetableoils, benzyl alcohols, polyetylene glycols, gelatine, carbohydrates suchas lactose, amylose or starch, magnesium stearate, talc, silicic acid,viscous paraffin, perfume oil, fatty acid monoglycerides anddiglycerides, pentaerythritol fatty acid esters, hydroxymethylcellulose, polyvinyl pyrrolidone, etc. Many more suitable carriersare described in Remmington's Pharmaceutical Sciences, 15th Edition,Easton:Mack Publishing Company, pages 1405-1412 and 1461-1487 (1975) andThe National Formulary XIV, 14th Edition, Washington, AmericanPharmaceutical Association (1975).

The gene constructs described herein, in particular, may be formulatedwith or administered in conjunction with agents that increase uptakeand/or expression of the gene construct by the cells relative to uptakeand/or expression of the gene construct by the cells that occurs whenthe identical genetic vaccine is administered in the absence of suchagents. Such agents and the protocols for administering them inconjunction with gene constructs are described in PCT Patent ApplicationSerial Number PCT/US94/00899 filed Jan. 26, 1994. Examples of suchagents include: CaPO₄, DEAE dextran, anionic lipids; extracellularmatrix-active enzymes; saponins; lectins; estrogenic compounds andsteroidal hormones; hydroxylated lower alkyls; dimethyl sulfoxide(DMSO); urea; and benzoic acid esters anilides, amidines, urethanes andthe hydrochloride salts thereof such as those of the family of localanesthetics. In addition, the gene constructs are encapsulatedwithin/administered in conjunction with lipids/polycationic complexes.

The compositions described herein can be sterilized and if desired mixedwith auxiliary agents, e.g., lubricants, preservatives, stabilizers,wetting agents, emulsifiers, salts for influencing osmotic pressure,buffers, coloring, flavoring and/or aromatic substances and the likethat do not deleteriously react with the adjuvant or the antigen.

The effective dose and method of administration of a particularformulation can vary based on the individual patient and the type andstage of the disease, as well as other factors known to those of skillin the art. Therapeutic efficacy and toxicity of the vaccines can bedetermined by standard pharmaceutical procedures in cell cultures orexperimental animals, e.g., ED₅₀ (the dose therapeutically effective in50% of the population). The data obtained from cell culture assays andanimal studies can be used to formulate a range of dosage for human use.The dosage of the vaccines lies preferably within a range of circulatingconcentrations that include the ED₅₀ with no toxicity. The dosage varieswithin this range depending upon the type of adjuvant derivative and HCVantigen, the dosage form employed, the sensitivity of the patient, andthe route of administration.

Since many adjuvants, including ribavirin, has been on the market forseveral years, many dosage forms and routes of administration are known.All known dosage forms and routes of administration can be providedwithin the context of the embodiments described herein. Preferably, anamount of adjuvant that is effective to enhance an immune response to anantigen in an animal can be considered to be an any amount that issufficient to achieve a blood serum level of antigen approximately0.25-12.5 μg/ml in the animal, preferably, about 2.5 μg/ml. In someembodiments, the amount of adjuvant is determined according to the bodyweight of the animal to be given the vaccine. Accordingly, the amount ofadjuvant in a particular formulation can be any amount between about0.1-6.0 mg/kg body weight. That is, some embodiments have an amount ofadjuvant that corresponds to any amount between 0.1-1.0 mg/kg, 1.1-2.0mg/kg, 2.1-3.0 mg/kg, 3.1-4.0 mg/kg, 4.1-5.0 mg/kg, 5.1, and 6.0 mg/kgbody weight of an animal. More conventionally, some of the compositionsdescribed herein contain any amount between about 0.25 mg-2000 mg ofadjuvant. That is, some embodiments have approximately 250 μg, 500 μg, 1mg, 25 mg, 50 mg, 100 mg, 150 mg, 200 mg, 250 mg, 300 mg, 350 mg, 400mg, 450 mg, 500 mg, 550 mg, 600 mg, 650 mg, 700 mg, 750 mg, 800 mg, 850mg, 900 mg, 1 g, 1.1 g, 1.2 g, 1.3 g, 1.4 g, 1.5 g, 1.6 g, 1.7 g, 1.8 g,1.9 g, and 2 g of adjuvant.

As one of skill in the art will appreciate, the amount of antigens in avaccine or immunogen preparation can vary depending on the type ofantigen and its immunogenicity. The amount of antigens in the vaccinescan vary accordingly. Nevertheless, as a general guide, the compositionsdescribed herein can have any amount between approximately 0.25-2000 mgof an HCV antigen discussed herein. For example, the amount of antigencan be between about 0.25 mg-5 mg, 5-10 mg, 10-100 mg, 100-500 mg, andupwards of 2000 mg. Preferably, the amount of HCV antigen is 0.1 μg-1mg, desirably, 1 μg-100 μg, preferably 5 μg-50 μg, and, most preferably,7 μg, 8 μg, 9 μg, 10 μg, 11 μg-20 μg, when said antigen is a nucleicacid and 1 μg-100 mg, desirably, 10 μg, preferably, 100 μg-1 mg, and,most preferably, 200 μg, 300 μg, 400 μg, 500 μg, 600 μg, or 700 μg-1 mg,when said antigen is a peptide.

In some approaches described herein, the exact amount of adjuvant and/orHCV antigen is chosen by the individual physician in view of the patientto be treated. Further, the amounts of adjuvant can be added incombination to or separately from the same or equivalent amount ofantigen and these amounts can be adjusted during a particularvaccination protocol so as to provide sufficient levels in light ofpatient-specific or antigen-specific considerations. In this vein,patient-specific and antigen-specific factors that can be taken intoaccount include, but are not limited to, the severity of the diseasestate of the patient, age, and weight of the patient, diet, time andfrequency of administration, drug combination(s), reactionsensitivities, and tolerance/response to therapy. The next sectiondescribes the use of ribavirin as an adjuvant in greater detail.

Ribavirin

Nucleoside analogs have been widely used in anti-viral therapies due totheir capacity to reduce viral replication. (Hosoya et al., J. Inf.Dis., 168:641-646 (1993)). ribavirin(1-β-D-ribofuranosyl-1,2,4-triazole-3-carboxamide) is a syntheticguanosine analog that has been used to inhibit RNA and DNA virusreplication. (Huffman et al., Antimicrob. Agents. Chemother., 3:235(1973); Sidwell et al., Science, 177:705 (1972)). Ribavirin has beenshown to be a competitive inhibitor of inositol mono-phosphate (IMP)dehydrogenase (IMPDH), which converts IMP to IMX (which is thenconverted to GMP). De Clercq, Anti viral Agents: characteristic activityspectrum depending on the molecular target with which they interact,Academic press, Inc., New York N.Y., pp. 1-55 (1993). Intracellularpools of GTP become depleted as a result of long term ribavirintreatment.

In addition to antiviral activity, investigators have observed that someguanosine analogs have an effect on the immune system. (U.S. Pat. Nos.6,063,772 and 4,950,647). Ribavirin has been shown to inhibit functionalhumoral immune responses (Peavy et al., J. Immunol., 126:861-864 (1981);Powers et al., Antimicrob. Agents. Chemother., 22:108-114 (1982)) andIgE-mediated modulation of mast cell secretion. (Marquardt et al., J.Pharmacol. Exp. Therapeutics, 240:145-149 (1987)). Some investigatorsreport that a daily oral therapy of ribavirin has an immune modulatingeffect on humans and mice. (Hultgren et al., J. Gen. Virol.,79:2381-2391 (1998) and Cramp et al., Gastron. Enterol., 118:346-355(2000)). Nevertheless, the current understanding of the effects ofribavirin on the immune system is in its infancy. As disclosed below,ribavirin was found to be a potent adjuvant.

Example 11

In a first set of experiments, groups of three to five Balb/c mice (BKUniversal, Uppsala, Sweden) were immunized i.p or s.c. (e.g., at thebase of the tail) with 10 μg or 100 μg of recombinant hepatitis C virusnon-structural 3 (rNS3) protein. The rNS3 was dissolved in phosphatebuffered saline (PBS) alone or PBS containing 1 mg ribavirin (obtainedfrom ICN, Costa Mesa, Calif.). Mice were injected with a total volume of100 μl per injection.

At two and four weeks following i.p. immunization, all mice were bled byretro-orbital sampling. Serum samples were collected and analyzed forthe presence of antibodies to rNS3. To determine the antibody titer, anenzyme immunoassay (EIA) was performed. (See e.g., Hultgren et al., JGen Virol. 79:2381-91 (1998) and Hultgren et al., Clin. Diagn. Lab.Immunol. 4:630-632 (1997)). The antibody levels were recorded as thehighest serum dilution giving an optical density at 405 nm more thantwice that of non-immunized mice.

Mice that received 10 μg or 100 μg rNS3 mixed with 1 mg ribavirin in PBSdisplayed consistently higher levels of NS3 antibodies. The antibodytiter that was detected by EIA at two weeks post-immunization is shownin FIG. 7. The vaccine formulations having 1 mg of ribavirin and either10 μg or 100 μg of rNS3 induced a significantly greater antibody titerthan the vaccine formulations composed of only rNS3.

In a second set of experiments, groups of eight Balb/c mice wereimmunized intraperitoneally with 10 or 50 μg of rNS3 in 100 μl phosphatebuffered saline containing either 0 mg, 1 mg, 3 mg, or 10 mg ribavirin(Sigma). At four, six and eight weeks the mice were bled and serum wasseparated and frozen. After completion of the study, sera were testedfor the levels of antibodies to recombinant NS3, as described above.Mean antibody levels to rNS3 were compared between the groups usingStudent's t-test (parametric analysis) or Mann-Whitney (non-parametricanalysis) and the software package StatView 4.5 (Abacus Concepts,Berkely, Calif.). The adjuvant effect of ribavirin when added in threedoses to 10 μg of rNS3 are provided in TABLE 11. The adjuvant effect ofribavirin when added in three doses to 50 μg of rNS3 are provided inTABLE 11. Parametrical comparison of the mean rNS3 antibody titres inmice receiving different 10 μg or 50 μg of rNS3 and different doses ofribavirin are provided in TABLES 12 and 13, respectively.Non-parametrical comparison of mean NS3 antibody titres in micereceiving different 10 μg or 50 μg of rNS3 and different doses ofribavirin are provided in TABLES 14-16, respectively. The values givenrepresent end point titres to recombinant rNS3.

TABLE 11 Amount Amount Antibody titre to rNS3 ribavirin immunogen atindicated week (mg/dose) (μg/dose) Mouse ID Week 4 Week 6 Week 8 None 105:1 300 1500 1500 None 10 5:2 <60 7500 1500 None 10 5:3 <60 1500 300None 10 5:4 60 1500 1500 None 10 5:5 <60 1500 nt None 10 5:6 60 15001500 None 10 5:7 <60 7500 7500 None 10 5:8 300 37500 7500 Group meantitre (mean ± SD) 180 ± 139  7500 ± 12421 3042 ± 3076 1 10 6:1 300 3750037500 1 10 6:2 <60 1500 1500 1 10 6:3 300 37500 187500 1 10 6:4 30037500 7500 1 10 6:5 60 nt nt 1 10 6:6 <60 37500 7500 1 10 6:7 <60 375007500 1 10 6:8 300 7500 7500 Group mean titre (mean ± SD) 252 ± 107 28071± 16195 36642 ± 67565 3 10 7:1 60 37500 7500 3 10 7:2 60 37500 37500 310 7:3 300 7500 7500 3 10 7:4 300 37500 7500 3 10 7:5 300 37500 37500 310 7:6 300 37500 37500 3 10 7:7 60 7500 7500 3 10 7:8 60 37500 37500Group mean titre (mean ± SD) 180 ± 128 30000 ± 13887 22500 ± 34637 10 108:1 300 37500 37500 10 10 8:2 300 37500 37500 10 10 8:3 <60 300 300 1010 8:4 60 7500 7500 10 10 8:5 <60 300 300 10 10 8:6 <60 37500 37500 1010 8:7 <60 7500 7500 10 10 8:8 <60 nt nt Group mean titre (mean ± SD)220 ± 139 18300 ± 18199 18300 ± 18199

TABLE 12 Amount Amount Antibody titre to rNS3 ribavirin immunogen atindicated week (mg/dose) (μg/dose) Mouse ID Week 4 Week 6 Week 8 None 501:1 60 7500 7500 None 50 1:2 60 7500 7500 None 50 1:3 60 7500 7500 None50 1:4 <60 1500 300 None 50 1:5 300 37500 37500 None 50 1:6 60 7500 7500None 50 1:7 60 37500 7500 None 50 1:8 Group mean titre (mean ± SD) 100 ±98  15214 ± 15380 10757 ± 12094 1 50 2:1 60 7500 7500 1 50 2:2 300 375007500 1 50 2:3 60 187500 7500 1 50 2:4 60 37500 187500 1 50 2:5 60 375007500 1 50 2:6 60 37500 37500 1 50 2:7 300 37500 7500 1 50 2:8 300 3750037500 Group mean titre (mean ± SD) 150 ± 124 52500 ± 55549 37500 ± 621053 50 3:1 60 37500 7500 3 50 3:2 300 37500 37500 3 50 3:3 300 37500 75003 50 3:4 60 37500 7500 3 50 3:5 300 37500 7500 3 50 3:6 60 37500 7500 350 3:7 — 7500 37500 3 50 3:8 1500 7500 37500 Group mean titre (mean ±SD) 387 ± 513 30000 ± 13887 18750 ± 15526 10 50 4:1 300 7500 7500 10 504:2 300 37500 37500 10 50 4:3 60 7500 7500 10 50 4:4 60 7500 7500 10 504:5 60 1500 1500 10 50 4:6 60 7500 37500 10 50 4:7 — 7500 7500 10 50 8:860 37500 7500 Group mean titre (mean ± SD) 140 ± 124 10929 ± 11928 15214± 15380

TABLE 13 Group Week Mean ± SD Group Mean ± SD analysis p-value 10 μgNS3/ 4 180 ± 139 10 μg NS3/ 252 ± 107 Students 0.4071 no ribavirin 1 mgribavirin t-test 6  7500 ± 12421 28071 ± 16195 Students 0.0156* t-test 83042 ± 3076 36642 ± 67565 Students 0.2133 t-test 10 μg NS3/ 4 180 ± 13910 μg NS3/ 180 ± 128 Students 1.000 no ribavirin 3 mg ribavirin t-test 6 7500 ± 12421 30000 ± 13887 Students 0.0042** t-test 8 3042 ± 3076 22500± 34637 Students 0.0077** t-test 10 μg NS3/ 4 180 ± 139 10 μg NS3/ 220 ±139 Students 0.7210 no ribavirin 10 mg ribavirin t-test 6  7500 ± 1242118300 ± 18199 Students 0.1974 t-test 8 3042 ± 3076 18300 ± 18199Students 0.0493* t-test

TABLE 14 Group Week Mean ± SD Group Mean ± SD analysis p-value 50 μgNS3/ 4 100 ± 98  50 μg NS3/ 150 ± 124 Students 0.4326 no ribavirin 1 mgribavirin t-test 6 15214 ± 15380 52500 ± 55549 Students 0.1106 t-test 810757 ± 12094 37500 ± 62105 Students 0.2847 t-test 50 μg NS3/ 4 100 ±98  50 μg NS3/ 387 ± 513 Students 0.2355 no ribavirin 3 mg ribavirint-test 6 15214 ± 15380 30000 ± 13887 Students 0.0721 t-test 8 10757 ±12094 18750 ± 15526 Students 0.2915 t-test 50 μg NS3/ 4 100 ± 98  50 μgNS3/ 140 ± 124 Students 0.5490 no ribavirin 10 mg ribavirin t-test 615214 ± 15380 10929 ± 11928 Students 0.5710 t-test 8 10757 ± 12094 15214± 15380 Students 0.5579 t-test Significance levels: NS = notsignificant; * = p < 0.05; ** = p < 0.01; *** = p < 0.001

TABLE 15 Group Week Mean ± SD Group Mean ± SD analysis p-value 10 μgNS3/ 4 180 ± 139 10 μg NS3/ 252 ± 107 Mann- 0.4280 no ribavirin 1 mgribavirin Whitney 6  7500 ± 12421 28071 ± 16195 Mann- 0.0253* Whitney 83042 ± 3076 36642 ± 67565 Mann- 0.0245* Whitney 10 μg NS3/ 4 180 ± 13910 μg NS3/ 180 ± 128 Mann- 0.0736 no ribavirin 3 mg ribavirin Whitney 6 7500 ± 12421 30000 ± 13887 Mann- 0.0050** Whitney 8 3042 ± 3076 22500 ±34637 Mann- 0.0034** Whitney 10 μg NS3/ 4 180 ± 139 10 μg NS3/ 220 ± 139Mann- 0.8986 no ribavirin 10 mg ribavirin Whitney 6  7500 ± 12421 18300± 18199 Mann- 0.4346 Whitney 8 3042 ± 3076 18300 ± 18199 Mann- 0.2102Whitney

TABLE 16 Group Week Mean ± SD Group Mean ± SD analysis p-value 50 μgNS3/ 4 100 ± 98  50 μg NS3/ 150 ± 124 Mann- 0.1128 no ribavirin 1 mgribavirin Whitney 6 15214 ± 15380 52500 ± 55549 Mann- 0.0210* Whitney 810757 ± 12094 37500 ± 62105 Mann- 0.1883 Whitney 50 μg NS3/ 4 100 ± 98 50 μg NS3/ 387 ± 513 Mann- 0.1400 no ribavirin 3 mg ribavirin Whitney 615214 ± 15380 30000 ± 13887 Mann- 0.0679 Whitney 8 10757 ± 12094 18750 ±15526 Mann- 0.2091 Whitney 50 μg NS3/ 4 100 ± 98  50 μg NS3/ 140 ± 124Mann- 0.4292 no ribavirin 10 mg ribavirin Whitney 6 15214 ± 15380 10929± 11928 Mann- 0.9473 Whitney 8 10757 ± 12094 15214 ± 15380 Mann- 0.6279Whitney Significance levels: NS = not significant; *= p < 0.05; **= p <0.01; ***= p < 0.001

I.

The data above demonstrates that ribavirin facilitates or enhances animmune response to an HCV antigen or HCV epitopes. A potent immuneresponse to rNS3 was elicited after immunization with a vaccinecomposition comprising as little as 1 mg ribavirin and 10 μg of rNS3antigen. The data above also provide evidence that the amount ofribavirin that is sufficient to facilitate an immune response to anantigen is between 1 and 3 mg per injection for a 25-30 g Balb/c mouse.It should be realized, however, that these amounts are intended forguidance only and should not be interpreted to limit the scope of theinvention in any way. Nevertheless, the data shows that vaccinecompositions comprising approximately 1 to 3 mg doses of ribavirininduce an immune response that is more than 12 times higher than theimmune response elicited in the absence of without ribavirin. Thus,ribavirin has a significant adjuvant effect on the humoral immuneresponse of an animal and thereby, enhances or facilitates the immuneresponse to the antigen. The example below describes experiments thatwere performed to better understand the amount of ribavirin needed toenhance or facilitate an immune response to an antigen.

Example 12

To determine a dose of ribavirin that is sufficient to provide anadjuvant effect, the following experiments were performed. In a firstset of experiments, groups of mice (three per group) were immunized witha 20 μg rNS3 alone or a mixture of 20 μg rNS3 and 0.1 mg, 1 mg, or 10 mgribavirin. The levels of antibody to the antigen were then determined byEIA. The mean endpoint titers at weeks 1 and 3 were plotted and areshown in FIG. 8. It was discovered that the adjuvant effect provided byribavirin had different kinetics depending on the dose of ribavirinprovided. For example, even low doses (<1 mg) of ribavirin were found toenhance antibody levels at week one but not at week three, whereas,higher doses (1-10 mg) were found to enhance antibody levels at weekthree.

A second set of experiments was also performed. In these experiments,groups of mice were injected with vaccine compositions comprisingvarious amounts of ribavirin and rNS3 and the IgG response in theseanimals was monitored. The vaccine compositions comprised approximately100 μl phosphate buffered saline and 20 μg rNS3 with or without 0.1 mg,1.0 mg, or 10 mg ribavirin (Sigma). The mice were bled at week six andrNS3-specific IgG levels were determined by EIA as described previously.As shown in TABLE 17, the adjuvant effects on the sustained antibodylevels were most obvious in the dose range of 1 to 10 mg per injectionfor a 25-30 g mouse.

TABLE 17 Amount (mg) ribavirin Endpoint titre of rNS3 IgG at mixed withindicated week Immunogen the immunogen Mouse ID Week 1 Week 2 Week 3 20μg rNS3 None 1 60 360 360 20 μg rNS3 None 2 360 360 2160 20 μg rNS3 None3 360 2160 2160 Mean 260 ± 173  960 ± 1039 1560 ± 1039 20 μg rNS3 0.1 42160 12960 2160 20 μg rNS3 0.1 5 60 60 60 20 μg rNS3 0.1 6 <60 2160 21601110 ± 1484 5060 ± 6921 1460 ± 1212 20 μg rNS3 1.0 7 <60 60 12960 20 μgrNS3 1.0 8 <60 2160 2160 20 μg rNS3 1.0 9 360 2160 2160 Mean 360 1460 ±1212 5760 ± 6235 20 μg rNS3 10.0 10 360 12960 77760 20 μg rNS3 10.0 11<60 2160 12960 20 μg rNS3 10.0 12 360 2160 2160 Mean 360 5760 ± 623530960 ± 40888

In a third set of experiments, the adjuvant effect of ribavirin afterprimary and booster injections was investigated. In these experiments,mice were given two intraperitoneal injections of a vaccine compositioncomprising 10 μg rNS3 with or without ribavirin and the IgG subclassresponses to the antigen was monitored, as before. Accordingly, micewere immunized with 100 μl phosphate buffered containing 10 recombinantNS3 alone, with or without 0.1 or 1.0 mg ribavirin (Sigma) at weeks 0and 4. The mice were bled at week six and NS3-specific IgG subclasseswere determined by EIA as described previously. As shown in TABLE 18,the addition of ribavirin to the immunogen prior to the injection doesnot change the IgG subclass response in the NS3-specific immuneresponse. Thus, the adjuvant effect of a vaccine composition comprisingribavirin and an antigen can not be explained by a shift in of theTh1/Th2-balance. It appears that another mechanism may be responsiblefor the adjuvant effect of ribavirin.

TABLE 18 Amount (mg) ribavirin Endpoint titre of indicated mixed withNS3 IgG subclass Immunogen the immunogen Mouse ID IgG1 IgG2a IgG2b IgG310 μg rNS3 None 1 360 60 <60 60 10 μg rNS3 None 2 360 <60 <60 60 10 μgrNS3 None 3 2160 60 <60 360 Mean 960 ± 1039 60 — 160 ± 173 10 μg rNS30.1 4 360 <60 <60 60 10 μg rNS3 0.1 5 60 <60 <60 <60 10 μg rNS3 0.1 62160 60 60 360 860 ± 1136 60 60 210 ± 212 10 μg rNS3 1.0 7 2160 <60 <6060 10 μg rNS3 1.0 8 360 <60 <60 <60 10 μg rNS3 1.0 9 2160 <60 <60 60Mean 1560 ± 1039  — — 60

The data presented in this example further verify that ribavirin can beadministered as an adjuvant and establish that that the dose ofribavirin can modulate the kinetics of the adjuvant effect. The examplebelow describes another assay that was performed to evaluate the abilityof ribavirin to enhance or facilitate an immune response to an antigen.

Example 13

This assay can be used with any ribavirin derivative or combinations ofribavirin derivatives to determine the extent that a particular vaccineformulation modulates a cellular immune response. To determine CD4⁺ Tcell responses to a ribavirin-containing vaccine, groups of mice wereimmunized s.c. with either 100 μg rNS3 in PBS or 100 μg rNS3 and 1 mgribavirin in PBS. The mice were sacrificed ten days post-immunizationand their lymph nodes were harvested and drained. In vitro recall assayswere then performed. (See e.g., Hultgren et al., J Gen Virol. 79:2381-91(1998) and Hultgren et al., Clin. Diagn. Lab. Immunol. 4:630-632(1997)). The amount of CD4⁺ T cell proliferation was determined at 96 hof culture by the incorporation of [³H] thymidine.

As shown in FIG. 9, mice that were immunized with 100 μg rNS3 mixed with1 mg ribavirin had a much greater T cell proliferative response thanmice that were immunized with 100 μg rNS3 in PBS. This data providesmore evidence that ribavirin enhances or facilitates a cellular immuneresponse (e.g., by promoting the effective priming of T cells).

Additional experiments were conducted to verify that ribavirin enhancesthe immune response to commercially available vaccine preparations. Theexample below describes the use of ribavirin in conjunction with acommercial HBV vaccine preparation.

Example 14

The adjuvant effect of ribavirin was tested when mixed with two doses ofa commercially available vaccine containing HBsAg and alum. (Engerix,SKB). Approximately 0.2 μg or 2 μg of Engerix vaccine was mixed witheither PBS or 1 mg ribavirin in PBS and the mixtures were injected intraperitoneally into groups of mice (three per group). A booster containingthe same mixture was given on week four and all mice were bled on weeksix. The serum samples were diluted from 1:60 to 1:37500 and thedilutions were tested by EIA, as described above, except that purifiedhuman HBsAg was used as the solid phase antigen. As shown in TABLE 19,vaccine formulations having ribavirin enhanced the response to 2 μg ofan existing vaccine despite the fact that the vaccine already containedalum. That is, by adding ribavirin to a suboptimal vaccine dose (i.e.,one that does not induce detectable antibodies alone) antibodies becamedetectable, providing evidence that the addition of ribavirin allows forthe use of lower antigen amounts in a vaccine formulation withoutcompromising the immune response.

TABLE 19 End point antibody titer to HBsAg in EIA 0.02 μg Engerix 0.2 μgEngerix No ribavirin 1 mg ribavirin No ribavirin 1 mg ribavirin Week #1#2 #3 #1 #2 #3 #1 #2 #3 #1 #2 #3 6 <60 <60 <60 <60 <60 <60 <60 <60 <60300 60 <60

The ribavirin used in the experiments above was obtained from commercialsuppliers (e.g., Sigma and ICN). The ribavirin that can be used with theembodiments described herein can also be obtained from commercialsuppliers or can be synthesized. The ribavirin and/or the antigen can beformulated with and without modification. For example, the ribavirin canbe modified or derivatized to make a more stable molecule and/or a morepotent adjuvant. By one approach, the stability of ribavirin can beenhanced by coupling the molecules to a support such as a hydrophilicpolymer (e.g., polyethylene glycol).

Many more ribavirin derivatives can be generated using conventionaltechniques in rational drug design and combinatorial chemistry. Forexample, Molecular Simulations Inc. (MSI), as well as many othersuppliers, provide software that allows one of skill to build acombinatorial library of organic molecules. The C2.Analog Builderprogram, for example, can be integrated with MSI's suite of Cerius2molecular diversity software to develop a library of ribavirinderivatives that can be used with the embodiments described herein. (Seee.g., http://msi.com/life/products/cerius2/index.html).

By one approach, the chemical structure of ribavirin is recorded on acomputer readable media and is accessed by one or more modeling softwareapplication programs. The C2.Analog Builder program in conjunction withC2-Diversity program allows the user to generate a very large virtuallibrary based on the diversity of R-groups for each substituentposition, for example. Compounds having the same structure as themodeled ribavirin derivatives created in the virtual library are thenmade using conventional chemistry or can be obtained from a commercialsource.

The newly manufactured ribavirin derivatives can then be screened inassays, which determine the extent of adjuvant activity of the moleculeand/or the extent of its ability to modulate of an immune response. Someassays may involve virtual drug screening software, such as C2.Ludi.C2.Ludi is a software program that allows a user to explore databases ofmolecules (e.g., ribavirin derivatives) for their ability to interactwith the active site of a protein of interest (e.g., RAC2 or another GTPbinding protein). Based upon predicted interactions discovered with thevirtual drug screening software, the ribavirin derivatives can beprioritized for further characterization in conventional assays thatdetermine adjuvant activity and/or the extent of a molecule to modulatean immune response. The section below provides more explanationconcerning the methods of using the compositions described herein.

Methods of Using the Vaccine Compositions and Immunogen Preparations

Routes of administration of the embodiments described herein include,but are not limited to, transdermal, parenteral, gastrointestinal,transbronchial, and transalveolar. Transdermal administration can beaccomplished by application of a cream, rinse, gel, etc. capable ofallowing the adjuvant and HCV antigen to penetrate the skin. Parenteralroutes of administration include, but are not limited to, electrical ordirect injection such as direct injection into a central venous line,intravenous, intramuscular, intraperitoneal, intradermal, orsubcutaneous injection. Gastrointestinal routes of administrationinclude, but are not limited to, ingestion and rectal. Transbronchialand transalveolar routes of administration include, but are not limitedto, inhalation, either via the mouth or intranasally.

Compositions having the adjuvant and HCV antigen that are suitable fortransdermal administration include, but are not limited to,pharmaceutically acceptable suspensions, oils, creams, and ointmentsapplied directly to the skin or incorporated into a protective carriersuch as a transdermal device (“transdermal patch”). Examples of suitablecreams, ointments, etc. can be found, for instance, in the Physician'sDesk Reference. Examples of suitable transdermal devices are described,for instance, in U.S. Pat. No. 4,818,540 issued Apr. 4, 1989 to Chinen,et al.

Compositions having the adjuvant and HCV antigen that are suitable forparenteral administration include, but are not limited to,pharmaceutically acceptable sterile isotonic solutions. Such solutionsinclude, but are not limited to, saline, phosphate buffered saline andoil preparations for injection into a central venous line, intravenous,intramuscular, intraperitoneal, intradermal, or subcutaneous injection.

Compositions having the adjuvant and HCV antigen that are suitable fortransbronchial and transalveolar administration include, but not limitedto, various types of aerosols for inhalation. Devices suitable fortransbronchial and transalveolar administration of these are alsoembodiments. Such devices include, but are not limited to, atomizers andvaporizers. Many forms of currently available atomizers and vaporizerscan be readily adapted to deliver vaccines having ribavirin and anantigen.

Compositions having the adjuvant and HCV antigen that are suitable forgastrointestinal administration include, but not limited to,pharmaceutically acceptable powders, pills or liquids for ingestion andsuppositories for rectal administration.

The gene constructs described herein, in particular, may be administeredby means including, but not limited to, traditional syringes, needlelessinjection devices, or “microprojectile bombardment gene guns”.Alternatively, the genetic vaccine may be introduced by various meansinto cells that are removed from the individual. Such means include, forexample, ex vivo transfection, electroporation, microinjection andmicroprojectile bombardment. After the gene construct is taken up by thecells, they are reimplanted into the individual. It is contemplated thatotherwise non-immunogenic cells that have gene constructs incorporatedtherein can be implanted into the individual even if the vaccinatedcells were originally taken from another individual.

According to some embodiments, the gene construct is administered to anindividual using a needleless injection device. According to someembodiments, the gene construct is simultaneously administered to anindividual intradermally, subcutaneously and intramuscularly using aneedleless injection device. Needleless injection devices are well knownand widely available. One having ordinary skill in the art can,following the teachings herein, use needleless injection devices todeliver genetic material to cells of an individual. Needleless injectiondevices are well suited to deliver genetic material to all tissue. Theyare particularly useful to deliver genetic material to skin and musclecells. In some embodiments, a needleless injection device may be used topropel a liquid that contains DNA molecules toward the surface of theindividual's skin. The liquid is propelled at a sufficient velocity suchthat upon impact with the skin the liquid penetrates the surface of theskin, permeates the skin and muscle tissue therebeneath. Thus, thegenetic material is simultaneously administered intradermally,subcutaneously and intramuscularly. In some embodiments, a needlelessinjection device may be used to deliver genetic material to tissue ofother organs in order to introduce a nucleic acid molecule to cells ofthat organ.

Preferred embodiments concern methods of treating or preventing HCVinfection. In these embodiments, an animal in need is provided an HCVantigen (e.g., a peptide antigen or nucleic acid-based antigen, asdescribed herein (SEQ. ID. NOs.: 1-27 and 35-36)) and an amount ofadjuvant sufficient to exhibit an adjuvant activity in said animal.Accordingly, an animal can be identified as one in need by usingcurrently available diagnostic testing or clinical evaluation. Theadjuvant and antigen can be provided separately or in combination, andother adjuvants (e.g., oil, alum, or other agents that enhance an immuneresponse) can also be provided to the animal in need.

Other embodiments of the invention include methods of enhancing animmune response to an HCV antigen by providing an animal in need with anamount of adjuvant (e.g., ribavirin) and one or more of SEQ. ID. NOs.:1-11 and 35-36, or a fragment thereof, preferably SEQ. ID. NOs.: 12-27that is effective to enhance said immune response. In these embodiments,an animal in need of an enhanced immune response to an antigen isidentified by using currently available diagnostic testing or clinicalevaluation. By one approach, for example, an uninfected individual isprovided with the vaccine compositions described above in an amountsufficient to elicit a cellular and humoral immune response to NS3 so asto protect said individual from becoming infected with HCV. In anotherembodiment, an HCV-infected individual is identified and provided with avaccine composition comprising ribavirin and NS3 in an amount sufficientto enhance the cellular and humoral immune response against NS3 so as toreduce or eliminate the HCV infection. Such individual may be in thechronic or acute phase of the infection. In yet another embodiment, anHCV-infected individual suffering from HCC is provided with acomposition comprising an adjuvant and the NS3/4A fusion gene in anamount sufficient to elicit a cellular and humoral immune responseagainst NS3-expressing tumor cells.

Although the invention has been described with reference to embodimentsand examples, it should be understood that various modifications can bemade without departing from the spirit of the invention. Accordingly,the invention is limited only by the following claims.

1. An immunogenic composition comprising a nucleic acid that comprises anucleotide sequence encoding a hepatitis C virus (HCV) NS3/4A fusionprotein in the absence of an additional HCV amino acid sequence, whereinsaid immunogenic composition is formulated for injection and, whereinsaid immunogenic composition induces the production of NS3 specificantibodies and NS3 specific T cells in mice that are immunized with saidimmunogenic composition.
 2. The immunogenic composition of claim 1,wherein said immunogenic composition induces production of a titer ofgreater than 10⁴ NS3 specific antibodies in immunized mice.
 3. Theimmunogenic composition of claim 1, wherein said immunogenic compositioninduces production of T cells that inhibit the growth of cells thatexpress NS3/4A in immunized mice.
 4. The immunogenic composition ofclaim 1, wherein said nucleotide sequence comprises at least 12consecutive nucleotides of SEQ. ID. NO.
 1. 5. The immunogeniccomposition of claim 1, wherein said nucleotide sequence comprises atleast 20 consecutive nucleotides of SEQ. ID. NO.
 1. 6. The immunogeniccomposition of claim 1, wherein said nucleotide sequence comprises atleast 20 consecutive nucleotides of SEQ. ID. NO.
 35. 7. The immunogeniccomposition of claim 1, wherein said nucleotide sequence comprises atleast 30 consecutive nucleotides of SEQ. ID. NO.
 35. 8. The immunogeniccomposition of claim 1, wherein said nucleotide sequence comprises atleast 50 consecutive nucleotides of SEQ. ID. NO.
 35. 9. The immunogeniccomposition of claim 1, wherein the nucleotide sequence encoding saidHCV NS3/4A fusion protein hybridizes to SEQ. ID. NO. 35 under stringentconditions.
 10. The immunogenic composition of claim 4, wherein saidnucleic acid comprises a nucleotide sequence comprising a substitutionof a different codon that encodes a functionally equivalent amino acid.11. The immunogenic composition of claim 8, wherein the nucleotidesequence encoding said HCV NS3/4A fusion protein hybridizes to SEQ. ID.NO. 35 under stringent conditions.
 12. The immunogenic composition ofclaim 1, wherein said NS3/4A fusion protein comprises a mutated NS3proteolytic cleavage site.
 13. The immunogenic composition of claim 5,wherein said NS3/4A fusion protein comprises a mutated NS3 proteolyticcleavage site.
 14. The immunogenic composition of claim 8, wherein saidNS3/4A fusion protein comprises a mutated NS3 proteolytic cleavage site.15. The immunogenic composition of claim 9, wherein said NS3/4A fusionprotein comprises a mutated NS3 proteolytic cleavage site.
 16. Theimmunogenic composition of claim 11, wherein said NS3/4A fusion proteincomprises a mutated NS3 proteolytic cleavage site.
 17. A mammalianexpression vector comprising: a nucleic acid that comprises a nucleotidesequence encoding a hepatitis C virus (HCV) NS3/4A fusion protein in theabsence of an additional HCV amino acid sequence, wherein said nucleicacid is operatively associated with at least one regulatory element thatdirects expression of said nucleic acid and, wherein said expressionvector induces production of NS3 specific antibodies and NS3 specific Tcells in mice that are immunized with said expression vector.
 18. Theexpression vector of claim 17, wherein said nucleotide sequencecomprises at least 20 consecutive nucleotides of SEQ. ID. NO.
 1. 19. Theexpression vector of claim 17, wherein said nucleotide sequencecomprises at least 50 consecutive nucleotides of SEQ. ID. NO.
 35. 20.The expression vector of claim 17, wherein the nucleotide sequenceencoding said HCV NS3/4A fusion protein hybridizes to SEQ. ID. NO. 35under stringent conditions.
 21. The expression vector of claim 19,wherein said nucleic acid hybridizes to SEQ. ID. NO. 35 under stringentconditions.
 22. The expression vector of claim 18, wherein said NS3/4Afusion protein comprises a mutated NS3 proteolytic cleavage site. 23.The expression vector of claim 21, wherein said NS3/4A fusion proteincomprises a mutated NS3 proteolytic cleavage site.
 24. The expressionvector of claim 17, wherein said at least one regulatory elementcomprises a promoter.
 25. The expression vector of claim 19, whereinsaid at least one regulatory element comprises an enhancer.